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http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:40:39Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
<span style="color: Blue">'''Whole-Cell PCR:'''</span><br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
<br />
<span style="color: Blue">'''Gel electrophoresis analysis and PCR product cleanup''':</span><br />
<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
<span style="color: Blue">'''PCR Purification : Easier protocol'''</span><br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
<span style="color: Blue">'''Plasmid miniprep''':</span><br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
<br />
<span style="color: Blue">'''Plasmid Gel Extraction:'''</span><br />
<br />
<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
<span style="color: Blue">'''Ligation'''</span><br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
<span style="color: Blue">'''Transformation by electroporation'''</span><br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:38:04Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
<span style="color: Blue">'''Whole-Cell PCR:'''</span><br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
<br />
<span style="color: Blue">'''Gel electrophoresis analysis and PCR product cleanup''':</span><br />
<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
<span style="color: Blue">'''PCR Purification : Easier protocol'''</span><br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
<span style="color: Blue">'''Plasmid miniprep''':</span><br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
<br />
<span style="color: Blue">Plasmid Gel Extraction:</span><br />
<br />
<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
<span style="color: Blue">'''Ligation'''</span><br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
<span style="color: Blue">'''Transformation by electroporation'''</span><br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:36:36Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
<span style="color: Blue">'''Whole-Cell PCR:'''</span><br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
<br />
<span style="color: Blue">'''Gel electrophoresis analysis and PCR product cleanup''':</span><br />
<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
<span style="color: Blue">'''PCR Purification : Easier protocol'''</span><br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
<span style="color: Blue">'''Plasmid miniprep''':</span><br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
<span style="color: Blue">'''Ligation'''</span><br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
<span style="color: Blue">'''Transformation by electroporation'''</span><br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:33:14Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
<span style="color: Blue">'''Whole-Cell PCR:'''</span><br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
<span style="color: Blue">'''Gel electrophoresis analysis and PCR product cleanup''':</span><br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:29:44Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
<span style="color: Blue">'''Whole-Cell PCR:'''</span><br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:26:28Z
<p>Rahula: </p>
<hr />
<div><span style="color: red"><div style="text-align: center;"><font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font></div></span><br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:15:03Z
<p>Rahula: </p>
<hr />
<div>:::::::<font size="5">'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''</font><br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T15:06:47Z
<p>Rahula: /* CLONING A CHROMOSOMAL GENE INTO A PLASMID */</p>
<hr />
<div>|align=center|==CLONING A CHROMOSOMAL GENE INTO A PLASMID==<br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T14:54:54Z
<p>Rahula: </p>
<hr />
<div>==CLONING A CHROMOSOMAL GENE INTO A PLASMID==<br />
<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
<br />
== Headline text ==</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T14:35:20Z
<p>Rahula: </p>
<hr />
<div>'''CLONING A CHROMOSOMAL GENE INTO A PLASMID'''<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T14:28:58Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tubes with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. Label the epi tubes in the mean time.<br />
# Dump elution from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min.<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-30T14:27:12Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
__________________________________________________________________________________________________________<br />
<br />
'''PCR Purification : Easier protocol'''<br />
# After PCR add Buffer PB in the ratio of 5:1 <br />
# Transfer to QIAGEN tuber with 10-100ul pipette. Label these.<br />
# Centrifuge for 1 min. label the epi tubes in the mean time.<br />
# Dump supernatant from collection tubes.<br />
# Elute with 750ul PE Buffer<br />
# Centrifuge for 1 min<br />
# Dump elution<br />
# Centrifuge again for 1 min<br />
# Put QIAspin columns into epi<br />
# Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.<br />
# Centrifuge again for 1 min<br />
__________________________________________________________________________________________________________<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:45:50Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
:2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:43:49Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
# Prepare Ligation Reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
2)Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes<br />
<br />
'''Transformation by electroporation'''<br />
<br />
# Thaw 100ul aliquots of cells from -80ºC.<br />
# Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.<br />
# Put the electroporation cuvettes on ice.<br />
# Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.<br />
# Pipette this mixture into a cuvette, inserting liquid between the 2 middle plates and tap down to ensure that it flows down. Set the electroporation pulser machine at 1450 mV<br />
# Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.<br />
# As soon as you hear a beep (which happens right away, 5ms, imperceptible length of time), pull out the cuvette, and add 1mL of SOC medium to the cuvette. ('''TIP: Rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants''') <br />
# Resuspend the “zapped” cells by pipetting up and down a few times.<br />
# Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used. <br />
# Incubate at 37ºC, shaking at 225 RPM. ('''TIP: Shaking may improve the recovery of transformants''')<br />
# Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent). <br />
<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:34:48Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:34:28Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:33:50Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
'''Ligation'''<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control'''(ul)||'''Ligation'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|Ligase Buffer|||2|||2<br />
|-<br />
|Plasmid|||5|||5<br />
|-<br />
|Insert|||-|||1.6<br />
|-<br />
|Ligase|||1|||1<br />
|-<br />
||||20|||20<br />
<br />
|} <br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_Gel_Extraction_2.jpg
File:BU Gel Extraction 2.jpg
2007-07-23T18:29:03Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:28:49Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
2) Carefully aspirate all supernatant media<br />
<br />
3) Put the eppy tube with the pelleted cell on ice<br />
<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg |center]]<br />
<br />
[[Image:BU_Gel Extraction 2.jpg |center]]<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:26:01Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg | center]]<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_Gel_Extraction_1.jpg
File:BU Gel Extraction 1.jpg
2007-07-23T18:24:12Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:23:54Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
[[Image:BU_Gel Extraction 1.jpg | center|]]<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T18:19:10Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''|||'''Control 1'''(ul)|||'''Control 2'''(ul)|||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T17:58:48Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''(ul)||'''Control 2'''(ul)||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
3) Run digestion for 90 minutes at 37 ºC<br />
<br />
Plasmid Gel Extraction:<br />
# Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer<br />
# Run the entire plasmid digestion product on the gel at 120V for 70 minutes<br />
# Use the orange transilluminator to visualize<br />
# With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band<br />
# Put the slice into a microcentrifuge tube<br />
# Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T17:55:52Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''(ul)||'''Control 2'''(ul)||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T17:55:12Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''(ul)||'''Control 2'''(ul)||'''PCR'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
Control 1 Control 2 Double Digestion<br />
Water 10.5 10.5 -<br />
EcoRI buffer 2 2 2<br />
BSA 2 2 2<br />
DNA 5 5 15<br />
EcoRI 0.5 - 0.5<br />
HindIII - 0.5 0.5<br />
20 µl 20 µl 20 µl<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T17:53:27Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
{| align="center" border="1"<br />
||||'''Control 1'''(ul)||'''Control 2'''(ul)||'''Double Digestion'''(ul)<br />
|-<br />
|Water|||10.5|||10.5||-<br />
|-<br />
|EcoRI Buffer1|||2|||2|||2<br />
|-<br />
|BSA|||2|||2||2<br />
|-<br />
|DNA|||5|||5|||15<br />
|-<br />
|EcoRI|||0.5|||-|||0.5<br />
|-<br />
|HindIII|||-|||0.5|||0.5<br />
|-<br />
||||20|||20|||2<br />
<br />
|} <br />
<br />
Control 1 Control 2 Double Digestion<br />
Water 10.5 10.5 -<br />
EcoRI buffer 2 2 2<br />
BSA 2 2 2<br />
DNA 5 5 15<br />
EcoRI 0.5 - 0.5<br />
HindIII - 0.5 0.5<br />
20 µl 20 µl 20 µl<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T16:44:06Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T16:43:26Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
<br />
<br />
<br />
[[Boston_University/Protocols | Back]]<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T16:42:19Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
''Note that these two steps should be done at the same time by different people''<br />
<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
<br />
50 ml TAE<br />
<br />
750 mg agarose<br />
<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
''Note that PCR product and vector digestion can be run simultaneously''<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_DNA_miniprep.jpg
File:BU DNA miniprep.jpg
2007-07-23T16:35:27Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T16:35:03Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
'''Plasmid miniprep''':<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA_miniprep.jpg|center|DNA Purification]]<br />
<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-23T16:27:18Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
<br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup <br />
<br />
'''Gel electrophoresis analysis and PCR product cleanup''':<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Image_Dump
Boston University/Image Dump
2007-07-23T15:21:07Z
<p>Rahula: </p>
<hr />
<div>[[Boston University | Back]]<br />
<br />
===ACHTUNG! The following page is merely a temporary storage space for images to be uploaded en masse before their use in specific pages. You may browse at your own peril.===<br />
<br />
[[Image:BU_lacz_PCR_cropped_7-17.jpg]]<br />
[[Image:BU_digested_pjq200_7-19-2007.jpg]]<br />
[[Image:BU_lacz_PCR_7-19-2007.jpg]]<br />
[[Image:BU_pjq200_sacb_digestion_7-17.jpg]]<br />
<br />
[[Image:mini_prep1.jpg]]<br />
[[Image:mini_prep2.jpg]]<br />
<br />
[[Image:tip_jar.jpg]]<br />
[[Image:thermocycler.jpg]]<br />
<br />
[[Image:electrophoresis1.jpg]]<br />
[[Image:electrophoresis2.jpg]]<br />
<br />
[[Image:shewy_shaker.jpg]]<br />
<br />
<br />
[[Image:BU_all_transforms.jpg]]<br />
[[Image:BU_dave_rahul_glove.jpg]]<br />
[[Image:BU_dave_rahul_holding_hands.jpg]]<br />
[[Image:BU_ecoli_zymo_electro.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad18s.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad18s_flash.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad30.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pjq200.jpg]]<br />
<br />
[[Image:BU_electro_pour_shewy.jpg]]<br />
[[Image:BU_electro_pour_shewy_2.jpg]]<br />
[[Image:BU_electro_pour_shewy_pbad18s.jpg]]<br />
[[Image:BU_electro_pour_shewy_pbad18s_bigger.jpg]]<br />
[[Image:BU_electro_pour_shewy_pjq200.jpg]]<br />
<br />
[[Image:BU_shewy_zymo_electro.jpg]]<br />
[[Image:BU_zymo_pour_ecoli.jpg]]<br />
[[Image:BU_zymo_pour_ecoli_pjq200.jpg]]<br />
[[Image:BU_zymo_pour_shewy.jpg]]<br />
[[Image:BU_zymo_streak_ecoli.jpg]]<br />
[[Image:BU_zymo_streak_shewy.jpg]]<br />
<br />
[[Image:BU_conjugation1.jpg]]<br />
[[Image:BU_conjugation2.jpg]]<br />
[[Image:BU_conjugation3.jpg]]<br />
[[Image:BU_conjugation4.jpg]]<br />
[[Image:BU_conjugation5.jpg]]<br />
<br />
[[Image:quia_king.jpg]]<br />
<br />
[[Image:restriction_tubes1.jpg]]<br />
[[Image:restriction_tubes2.jpg]]<br />
<br />
[[Image:nano_drop1.jpg]]<br />
[[Image:nano_drop2.jpg]]<br />
<br />
[[Image:gel_microwave.jpg]]<br />
<br />
[[Image:ecoli_shaker.jpg]]<br />
[[Image:ecoli_incubator.jpg]]<br />
[[Image:ecoli_incubator.jpg]]<br />
<br />
[[Image:frozen_stocks1.jpg]]<br />
[[Image:frozen_stocks2.jpg]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Image_Dump
Boston University/Image Dump
2007-07-23T15:12:01Z
<p>Rahula: </p>
<hr />
<div>[[Boston University | Back]]<br />
<br />
===ACHTUNG! The following page is merely a temporary storage space for images to be uploaded en masse before their use in specific pages. You may browse at your own peril.===<br />
<br />
[[Image:BU_lacz_PCR_cropped_7-17.jpg]]<br />
[[Image:BU_digested_pjq200_7-19-2007.jpg]]<br />
[[Image:BU_lacz_PCR_7-19-2007.jpg]]<br />
[[Image:BU_pjq200_sacb_digestion_7-17.jpg]]<br />
<br />
<br />
<br />
[[Image:BU_all_transforms.jpg]]<br />
[[Image:BU_dave_rahul_glove.jpg]]<br />
[[Image:BU_dave_rahul_holding_hands.jpg]]<br />
[[Image:BU_ecoli_zymo_electro.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad18s.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad18s_flash.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pbad30.jpg]]<br />
[[Image:BU_electro_pour_ecoli_pjq200.jpg]]<br />
<br />
[[Image:BU_electro_pour_shewy.jpg]]<br />
[[Image:BU_electro_pour_shewy_2.jpg]]<br />
[[Image:BU_electro_pour_shewy_pbad18s.jpg]]<br />
[[Image:BU_electro_pour_shewy_pbad18s_bigger.jpg]]<br />
[[Image:BU_electro_pour_shewy_pjq200.jpg]]<br />
<br />
[[Image:BU_shewy_zymo_electro.jpg]]<br />
[[Image:BU_zymo_pour_ecoli.jpg]]<br />
[[Image:BU_zymo_pour_ecoli_pjq200.jpg]]<br />
[[Image:BU_zymo_pour_shewy.jpg]]<br />
[[Image:BU_zymo_streak_ecoli.jpg]]<br />
[[Image:BU_zymo_streak_shewy.jpg]]<br />
<br />
[[Image:BU_conjugation1.jpg]]<br />
[[Image:BU_conjugation2.jpg]]<br />
[[Image:BU_conjugation3.jpg]]<br />
[[Image:BU_conjugation4.jpg]]<br />
[[Image:BU_conjugation5.jpg]]<br />
<br />
[[Image:quia_king.jpg]]<br />
<br />
[[Image:restriction_tubes1.jpg]]<br />
[[Image:restriction_tubes2.jpg]]<br />
<br />
[[Image:nano_drop1.jpg]]<br />
[[Image:nano_drop2.jpg]]<br />
<br />
[[Image:gel_microwave.jpg]]<br />
<br />
[[Image:ecoli_shaker.jpg]]<br />
[[Image:ecoli_incubator.jpg]]<br />
[[Image:ecoli_incubator.jpg]]<br />
<br />
[[Image:frozen_stocks1.jpg]]<br />
[[Image:frozen_stocks2.jpg]]<br />
<br />
[[Image:mini_prep1.jpg]]<br />
[[Image:mini_prep2.jpg]]<br />
<br />
[[Image:tip_jar.jpg]]<br />
[[Image:thermocycler.jpg]]<br />
<br />
[[Image:electrophoresis1.jpg]]<br />
[[Image:electrophoresis2.jpg]]<br />
<br />
[[Image:shewy_shaker.jpg]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Gel_Protocol
Boston University/Gel Protocol
2007-07-23T15:04:41Z
<p>Rahula: /* Making Electrophoresis Gel */</p>
<hr />
<div>== Making Electrophoresis Gel ==<br />
<br />
# Take 60ml TAE 1X and add 0.9g agarose to make a 1.5% agarose gel.<br />
# Microwave for about 1-1.5 minutes and shake after every 20 seconds. Make SURE that all the agarose is COMPLETELY dissolved (the solution should be clear).<br />
# Let the solution cool by placing the flask under running tap water for about a minute to a minute and a half. No water should get inside the flask. Make sure that you don't cool the gel flask too much otherwise it will solidify. <br />
# Add 1ul of Etbr and swirl around. <br />
# Pour it on the gel-plate with a fork already in it.<br />
<br />
'''Loading'''<br />
<br />
Load 10ul of diluted ladder in a lane.<br />
<br />
The Fisher 1/40loading dye should have a final volume 1/5 in the overall solution.<br />
<br />
[[Boston_University/Protocols | Back]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T18:36:21Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T18:36:04Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|''''''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
||||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T16:27:10Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
<br />
:The contents of the plates are as follows(column indicates transformation type): <br />
<br />
{| align="center" border="1"<br />
|'''::::::'''||'''Control 1'''||'''Control 2'''||'''PCR'''<br />
|-<br />
|Water|||10.5|||10.5||8.5<br />
|-<br />
|Primer 1|||2|||-|||2<br />
|-<br />
|Primer 2|||-|||2||2<br />
|-<br />
|Master Mix|||12.5|||12.5|||12.5<br />
|-<br />
|::::|||25|||25|||25<br />
<br />
|}<br />
<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:37:35Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]<br />
<br />
Note that PCR product and vector digestion can be run simultaneously<br />
<br />
PCR product digestion:<br />
1) Prepare 20 µl digestion reaction:<br />
2 µl EcoRI buffer<br />
2 µl BSA<br />
15 µl DNA<br />
0.5 µl EcoRI<br />
0.5 µl HindIII<br />
2) Run digestion for 90 minutes at 37ºC<br />
<br />
Clean up:<br />
1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl<br />
<br />
<br />
Plasmid digestion:<br />
1) Use miniprepped plasmid<br />
2) Prepare plasmid digestion reaction</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_DNA_Purification3.jpg
File:BU DNA Purification3.jpg
2007-07-20T15:34:59Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:34:47Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
[[Image:BU_DNA Purification3.jpg|center|DNA Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:31:40Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:28:55Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]<br />
<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_DNA_Purification2.jpg
File:BU DNA Purification2.jpg
2007-07-20T15:22:36Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:20:47Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg |center| PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_DNA_Purification.jpg
File:BU DNA Purification.jpg
2007-07-20T15:19:56Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:18:40Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg | PCR Purification]]<br />
<br />
3) Elute with 35 µl EB instead of 50 µl<br />
4) Measure concentration with the Nanodrop<br />
<br />
<br />
<br />
Plasmid miniprep:<br />
<br />
1) Miniprep using a Qiagen Miniprep Kit:<br />
<br />
[[Image:BU_DNA Purification.jpg|center|DNA Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/File:BU_PCR_Purification.jpg
File:BU PCR Purification.jpg
2007-07-20T15:05:01Z
<p>Rahula: </p>
<hr />
<div></div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:04:52Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR Purification.jpg | PCR Purification]]</div>
Rahula
http://2007.igem.org/wiki/index.php/Boston_University/Custom_Cloning_Protocol
Boston University/Custom Cloning Protocol
2007-07-20T15:02:41Z
<p>Rahula: </p>
<hr />
<div>Cloning a chromosomal gene into a plasmid<br />
<br />
Whole-Cell PCR (3 hours)<br />
-prepare agarose gel<br />
Gel electrophoresis with SYBR safe (1 hour)<br />
-prepare Qiagen PCR Cleanup Kit<br />
Qiagen PCR Cleanup (0.5 hour)<br />
-prepare digestion plasmid<br />
Prepare digestion of PCR products (0.5 hour)<br />
Digestion of PCR products and plasmid (3 hours)<br />
-prepare Qiagen PCR Cleanup Kit<br />
-prepare agarose gel (SYBR safe)<br />
Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)<br />
-prepare Qiagen Gel Extraction Kit<br />
Qiagen Gel Extraction of plasmid from gel (0.5 hour)<br />
-prepare ligation<br />
Ligation (1 hour)<br />
-prepare competent cells from O/N culture<br />
Transformation (0.5 hour)<br />
-warm plates<br />
Plate cells<br />
<br />
<br />
Whole-Cell PCR:<br />
<br />
1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube<br />
2) Carefully aspirate all supernatant media<br />
3) Put the eppy tube with the pelleted cell on ice<br />
4) Mix the 25 µl PCR cocktail:<br />
<br />
Control 1 Control 2 PCR<br />
Water 10.5 10.5 8.5<br />
Primer 1 2 - 2<br />
Primer 2 - 2 2<br />
Master Mix 12.5 12.5 12.5<br />
25 µl 25 µl 25 µl<br />
5)Thermocycler<br />
94ºC 5 min <br />
94ºC 30 sec <------ <br />
55ºC 1 min | repeat 35 X <br />
65ºC 4 min <------ <br />
65ºC 10 min <br />
6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)<br />
<br />
<br />
Note that these two steps should be done at the same time by different people<br />
1) Run 5µl of PCR product in a 1.5% agarose gel<br />
50 ml TAE<br />
750 mg agarose<br />
0.5 µl 1% EtBr<br />
<br />
120V for 45 minutes<br />
<br />
2) Clean up rest of PCR product with a Qiagen PCR cleanup kit <br />
<br />
[[Image:BU_PCR_Purification | PCR Purification.jpg]]</div>
Rahula