From 2007.igem.org

CLONING A CHROMOSOMAL GENE INTO A PLASMID

Whole-Cell PCR (3 hours) -prepare agarose gel Gel electrophoresis with SYBR safe (1 hour) -prepare Qiagen PCR Cleanup Kit Qiagen PCR Cleanup (0.5 hour) -prepare digestion plasmid Prepare digestion of PCR products (0.5 hour) Digestion of PCR products and plasmid (3 hours) -prepare Qiagen PCR Cleanup Kit -prepare agarose gel (SYBR safe) Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour) -prepare Qiagen Gel Extraction Kit Qiagen Gel Extraction of plasmid from gel (0.5 hour) -prepare ligation Ligation (1 hour) -prepare competent cells from O/N culture Transformation (0.5 hour) -warm plates Plate cells

Whole-Cell PCR:

1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube

2) Carefully aspirate all supernatant media

3) Put the eppy tube with the pelleted cell on ice

4) Mix the 25 µl PCR cocktail:

The contents of the plates are as follows(column indicates transformation type):

	Control 1'(ul)	Control 2(ul)	PCR(ul)
Water	10.5	10.5	8.5
Primer 1	2	-	2
Primer 2	-	2	2
Master Mix	12.5	12.5	12.5
	25	25	25

5)Thermocycler 94ºC 5 min 94ºC 30 sec <------ 55ºC 1 min | repeat 35 X 65ºC 4 min <------ 65ºC 10 min

6) Prepare Gel electrophoresis analysis and PCR product cleanup

Gel electrophoresis analysis and PCR product cleanup:

Note that these two steps should be done at the same time by different people

1) Run 5µl of PCR product in a 1.5% agarose gel

50 ml TAE

750 mg agarose

0.5 µl 1% EtBr

120V for 45 minutes

2) Clean up rest of PCR product with a Qiagen PCR cleanup kit

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PCR Purification : Easier protocol

1. After PCR add Buffer PB in the ratio of 5:1
2. Transfer to QIAGEN tubes with 10-100ul pipette. Label these.
3. Centrifuge for 1 min. Label the epi tubes in the mean time.
4. Dump elution from collection tubes.
5. Elute with 750ul PE Buffer
6. Centrifuge for 1 min
7. Dump elution
8. Centrifuge again for 1 min
9. Put QIAspin columns into epi
10. Elute with 30-35ul Buffer EB. Make sure the EB fall directly in the middle of the filter.
11. Centrifuge again for 1 min.

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3) Elute with 35 µl EB instead of 50 µl

4) Measure concentration with the Nanodrop

Plasmid miniprep:

1) Miniprep using a Qiagen Miniprep Kit:

Note that PCR product and vector digestion can be run simultaneously

PCR product digestion: 1) Prepare 20 µl digestion reaction: 2 µl EcoRI buffer 2 µl BSA 15 µl DNA 0.5 µl EcoRI 0.5 µl HindIII 2) Run digestion for 90 minutes at 37ºC

Clean up: 1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl

Plasmid digestion: 1) Use miniprepped plasmid 2) Prepare plasmid digestion reaction

	Control 1(ul)	Control 2(ul)	Double Digestion(ul)
Water	10.5	10.5
EcoRI Buffer1	2	2	2
BSA	2	2	2
DNA	5	5	15
EcoRI	0.5	-	0.5
HindIII	-	0.5	0.5
	20	20	2

3) Run digestion for 90 minutes at 37 ºC

Plasmid Gel Extraction:

1. Prepare a 60ml 1% agarose gel with SYBR Safe (or Gold) as a stainer
2. Run the entire plasmid digestion product on the gel at 120V for 70 minutes
3. Use the orange transilluminator to visualize
4. With a scalpel or razor blade, cut a thin slice of gel containing the double-digested DNA band
5. Put the slice into a microcentrifuge tube
6. Use the Qiagen Gel Extraction Kit to clean the DNA (Do not use the second QG wash and elute with 35 µl)

Ligation

1. Prepare Ligation Reaction

	Control(ul)	Ligation(ul)
Water	10.5	10.5
Ligase Buffer	2	2
Plasmid	5	5
Insert	-	1.6
Ligase	1	1
	20	20

2. Place ligation reaction tubes in the thermocycler at 16 ºC for 30 minutes

Transformation by electroporation

1. Thaw 100ul aliquots of cells from -80ºC.
2. Have plasmids ready. Just need 1 µl of miniprepped (usually 20-30 ng/µl) plasmid. Use at least 10 ng of plasmid DNA.
3. Put the electroporation cuvettes on ice.
4. Once the cells are thawed, add 50 µl of cells + 1 µl of DNA and vortex/mix in an eppy tube.
5. 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
6. Push the cuvette (with the tab in the front) into its slot in the machine and hit the yellow Start button.
7. 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)
8. Resuspend the “zapped” cells by pipetting up and down a few times.
9. Transfer the ~ 1mL of cells in SOC into the original Eppy tubes used.
10. Incubate at 37ºC, shaking at 225 RPM. (TIP: Shaking may improve the recovery of transformants)
11. Plate low-copy plasmid-containing transformed cells onto 7.5µg/ml LB-Gent plates. (If E.Coli use higher concerntration of Gent).

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