From 2007.igem.org

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Revision as of 06:13, 26 October 2007

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