Berkeley LBL/Mimi-SchlI

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Revision as of 19:28, 26 October 2007

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 by DNA 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

12. Transformation into BL21 cells via KCM Competent Cell Transformation:

13. Protein Expression

14. Protein Analysis using SDS-PAGE