Berkeley LBL/MimiNotebook

From 2007.igem.org

(Difference between revisions)
Line 23: Line 23:
-
'''pET3A-(S)-chlH:'''
+
'''Construction of ''pET3A-(S)-chlH'':'''
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
1. Amplify Synechocystis-Cyanobacteria gene ''S-chlH'' by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
-
Amplification introduces restriction sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
+
Amplification introduces sites ''NdeI'' and ''KpnI-BamHI'' into the gene.
-
2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene.
+
2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
-
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of plasmid ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
+
4. [[Berkeley_LBL/Digestion|Restriction Digestion]] of gene ''S-chlH'' with ''NdeI'' and ''BamHI'' using the following conditions:
Line 46: Line 46:
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'''"
 +
 +
8. Transformation into ''Novablue'' cells using [[Berkeley_LBL/CompetentCellTransformation|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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
 +
 +
11. [[Berkeley_LBL/Digestion2|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 [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]] using the following conditions:
 +
 +
 +
Amplification introduces sites ''KpnI-rbs'' and ''SpeI-NotI-BglII'' into the gene.
 +
 +
2. Amplification is followed by[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]] is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~1kb).
 +
 +
3. [[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]
 +
 +
4. [[Berkeley_LBL/Digestion|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. [[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.
 +
 +
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:
 +
 +
 +
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. [[Berkeley_LBL/Miniprep|Miniprep]] cultures
 +
 +
11. [[Berkeley_LBL/Digestion2|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

Revision as of 05:29, 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 enzymesNdeI and BamHIusing 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


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.

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

6. Gel Extraction is performed to isolate the correct band.

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

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

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