Berkeley LBL/MimiNotebook

From 2007.igem.org

(Difference between revisions)
Line 14: Line 14:
2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.
2. [[Berkeley_LBL/Miniprep|Miniprep]] cultures.
-
3. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes''NdeI'' and ''BamHI''using the following conditions:
+
3. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A'' with enzymes ''NdeI'' and ''BamHI'' using the following conditions:
         42.1 ul pET3A plasmid
         42.1 ul pET3A plasmid
Line 21: Line 21:
         1.2 ul NdeI
         1.2 ul NdeI
         1.2 ul BamHI
         1.2 ul BamHI
 +
        ---------------------
 +
        50 ul total
 +
 +
4. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~5kb).
   
   
Line 38: Line 42:
Digest in 37°C for 2 hours.
Digest in 37°C for 2 hours.
 +
Add 0.5 ul NdeI and 0.5 ul BamHI.
Add 0.5 ul NdeI and 0.5 ul BamHI.
 +
Digest in 37°C for additional 30 minutes.
Digest in 37°C for additional 30 minutes.
5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band.
+
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct band(~4kb).
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlH'' to plasmid ''pET3A", yielding plasmid "'''pET3A-(S)-chlH'''"
Line 87: Line 93:
Digest in 37°C for 2 hours.
Digest in 37°C for 2 hours.
 +
Add 0.5 ul KpnI and 0.5 ul BglII.
Add 0.5 ul KpnI and 0.5 ul BglII.
 +
Digest in 37°C for additional 30 minutes.
Digest in 37°C for additional 30 minutes.
-
5. [[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm the correct band of ~4kb.
+
5. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlH'' with ''KpnI'' and ''BamHI'' using the following conditions:
-
 
+
-
6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlH'' with ''KpnI'' and ''BamHI'' using the following conditions:
+
Digest in 37°C for 2 hours.
Digest in 37°C for 2 hours.
 +
Add 0.5 ul KpnI and 0.5 ul BglII.
Add 0.5 ul KpnI and 0.5 ul BglII.
 +
Digest in 37°C for additional 30 minutes.
Digest in 37°C for additional 30 minutes.
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions.
+
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).
 +
 
 +
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''" using the following conditions:
-
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlI'' to plasmid ''pET3A-(S)-chlH", yielding plasmid "'''pET3A-(S)-chlHI'''"
 
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
Line 170: Line 179:
2 hour digestion in 37°C
2 hour digestion in 37°C
 +
Add 0.5 ul SpeI
Add 0.5 ul SpeI
 +
30 min digestion in 37°C
30 min digestion in 37°C
 +
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
Line 181: Line 193:
2 hour digestion in 37°C
2 hour digestion in 37°C
 +
Add 0.5 ul NotI
Add 0.5 ul NotI
 +
30 min digestion in 37°C
30 min digestion in 37°C
5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker
5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker
-
[[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion.
 
-
6. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlHI'' with ''SpeI'' and ''NotI'' using the following conditions:
+
6. [[Berkeley_LBL/Miniprep|Miniprep]] cultures and prepare for digestion.
 +
 
 +
7. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''pET3A-(S)-schlHI'' with ''SpeI'' and ''NotI'' using the following conditions:
''Sequential Restriction Digestion for pEt3A-(S)-HI:''
''Sequential Restriction Digestion for pEt3A-(S)-HI:''
Line 198: Line 213:
2 hour digestion in 37°C
2 hour digestion in 37°C
 +
Add 0.5 ul SpeI
Add 0.5 ul SpeI
 +
30 min digestion in 37°C
30 min digestion in 37°C
 +
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
[[Berkeley_LBL/PCRcleanup|Clean Up/Purification]]
Line 209: Line 227:
2 hour digestion in 37°C
2 hour digestion in 37°C
 +
Add 0.5 ul NotI
Add 0.5 ul NotI
 +
30 min digestion in 37°C
30 min digestion in 37°C
-
6. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
+
8. [[Berkeley_LBL/GelExtraction|Gel Extraction]] is performed to isolate the correct bands for both digestions (~2kb and ~10kb).
-
7. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:
+
9. [[Berkeley_LBL/Ligation|Ligate]] ''S-chlD'' to plasmid ''pET3A-(S)-chlHI", yielding plasmid "'''pET3A-(S)-chlHID'''" using the following conditions:
               12 ul pET3A-(S)-HI
               12 ul pET3A-(S)-HI
Line 224: Line 244:
               20 ul total
               20 ul total
-
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
+
10. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]] using the following conditions:
               7 ul pET3A-(S)-HID ligation
               7 ul pET3A-(S)-HID ligation

Revision as of 06:09, 26 October 2007

Contruction of pET3a Derivatives Containing S-chlH, S-chlI, and S-chlD

Cyanobacteria - Synechocystis 

       S-chlH: 3996 base pairs
       S-chlI: 1074 base pairs
       S-chlD: 2031 base pairs

Sequences and Properties of Oligonucleotides

pET3A:

1. Innoculate pET3A single colony from plate into 250 mL flask containing 50 mL LB + 50 ul Carb. Allow to grow in 30°C shaker overnight.

2. Miniprep cultures.

3. Restriction Digestion of plasmid pET3A with enzymes NdeI and BamHI using the following conditions: 

        42.1 ul pET3A plasmid
        5 ul NEB 4 (10x)
        0.5 ul BSA (100x)
        1.2 ul NdeI
        1.2 ul BamHI
        ---------------------
        50 ul total

4. Gel Extraction is performed to isolate the correct band(~5kb).


Construction of pET3A-(S)-chlH:

1. Amplify Synechocystis-Cyanobacteria gene S-chlH by PCR (Using Phusion Polymerase) using the following conditions:


Amplification introduces sites NdeI and KpnI-BamHI into the gene.

2. Amplification is followed byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlH with NdeI and BamHI using the following conditions:


Digest in 37°C for 2 hours.

Add 0.5 ul NdeI and 0.5 ul BamHI.

Digest in 37°C for additional 30 minutes.

5. DNA Gel Electrophoresis is used to confirm the correct band of ~4kb.

6. Gel Extraction is performed to isolate the correct band(~4kb).

7. Ligate S-chlH to plasmid pET3A", yielding plasmid "pET3A-(S)-chlH"

8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:


Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions: 

           20 ul DNA
           3 ul NEB 4 (10x)
           1 ul NdeI
           1 ul SpeI
           3 ul BSA (10x)
           2.0 ul H2O   
           -------------
           30 ul total

Run gel – look for ~4kb and ~5kb band

Save glycerol stocks


Construction of pET3A-(S)-chlHI:

1. Amplify Synechocystis-Cyanobacteria gene S-chlI by PCR (Using Phusion Polymerase) using the following conditions:


Amplification introduces sites KpnI-rbs and SpeI-NotI-BglII into the gene.

2. Amplification is followed byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~1kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlI with KpnI and BglII using the following conditions:


Digest in 37°C for 2 hours.

Add 0.5 ul KpnI and 0.5 ul BglII.

Digest in 37°C for additional 30 minutes.

5. Restriction Digestion of plasmid pET3A-(S)-schlH with KpnI and BamHI using the following conditions:


Digest in 37°C for 2 hours.

Add 0.5 ul KpnI and 0.5 ul BglII.

Digest in 37°C for additional 30 minutes.

6. Gel Extraction is performed to isolate the correct bands for both digestions (~1kb for S-chlI and ~9kb for pET3A-(S)-chlH).

7. Ligate S-chlI to plasmid pET3A-(S)-chlH", yielding plasmid "pET3A-(S)-chlHI" using the following conditions:


8. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:


Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions: 

           20 ul DNA
           3 ul NEB 1 (10x)
           1.4 ul SpeI
           0.9 ul KpnI
           3 ul BSA (10x)
           1.7 ul H2O   
           -------------
           30 ul total

Run gel – look for ~1kb and ~9kb band

Save glycerol stocks


Construction of pET3A-(S)-chlHID:

1. Amplify Synechocystis-Cyanobacteria gene S-chlD by PCR (Using Phusion Polymerase) using the following conditions: 

           PCR:
           1 ul Schl-D
           10 ul HF Buffer 5x
           1 ul dNTP
           5 ul primer mix
           0.5 ul Phusion
           32.5 ul H2O
           --------------
           50 ul total

           Conditions:
           98°C	30s
           98°C 	8s
           61°C	30s
           72°C	1:10m
           Go to 2 for additional 29 cycles
           72°C	10m
           4°C	        ---


Amplification introduces sites SpeI-rbs and NotI-BglII into the gene.

2. Amplification is followed byDNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~2kb).

3. PCR Clean Up/Purification

4. Restriction Digestion of gene S-chlD with SpeI and NotI using the following conditions:

Schl-D Sequential Restriction Digestion: 

              Digestion #1
              43 ul Schl-D
              5 ul NEB 2 (10x)
              0.5 ul BSA (100x)
              1.5 ul SpeI

2 hour digestion in 37°C

Add 0.5 ul SpeI

30 min digestion in 37°C

Clean Up/Purification 

              Digestion #2
              43 ul Schl-D
              5 ul NEB 3 (10x)
              0.5 ul BSA (100x)
              1.5 ul NotI

2 hour digestion in 37°C

Add 0.5 ul NotI

30 min digestion in 37°C

5. Added a scrape of pET3A-(S)-HI glycerol stock to 4 mL LB + 4 mL Carb and let grow overnight in 30°C shaker

6. Miniprep cultures and prepare for digestion.

7. Restriction Digestion of plasmid pET3A-(S)-schlHI with SpeI and NotI using the following conditions:

Sequential Restriction Digestion for pEt3A-(S)-HI: 

              Digestion #1:
              43 ul pEt3A-(S)-HI
              5 ul NEB 2 (10x)
              0.5 ul BSA (100x)
              1.5 ul SpeI

2 hour digestion in 37°C

Add 0.5 ul SpeI

30 min digestion in 37°C

Clean Up/Purification 

              Digestion #2:
              43 ul pEt3A-(S)-HI
              5 ul NEB 3 (10x)
              0.5 ul BSA (100x)
              1.5 ul NotI

2 hour digestion in 37°C

Add 0.5 ul NotI

30 min digestion in 37°C

8. Gel Extraction is performed to isolate the correct bands for both digestions (~2kb and ~10kb).

9. Ligate S-chlD to plasmid pET3A-(S)-chlHI", yielding plasmid "pET3A-(S)-chlHID" using the following conditions: 

             12 ul pET3A-(S)-HI
             4 ul Schl-D
             2 ul Ligase Buffer
             1 ul Ligase Enzyme
             1 ul H2O        
             -------------------
             20 ul total

10. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions: 

             7 ul pET3A-(S)-HID ligation
             73 ul H2O
             20 ul KCM solution
             100 ul Chemical Competent Novablue cells
             -----------------------------------------
             200 ul total

Plate onto LB Agar + Carb plate

9. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.

10. Miniprep cultures

11. Analytic Digestion using the following conditions: 

           20 ul DNA
           3 ul NEB 2 (10x)
           1.8 ul NotI
           1 ul SpeI
           3 ul BSA (10x)
           1.2 ul H2O  
           -------------
           30 ul total

Run gel – look for ~2kb and ~9kb band

Save glycerol stocks

Retrieved from "http://2007.igem.org/wiki/index.php/Berkeley_LBL/MimiNotebook"