Berkeley LBL/Mimi-SchlI
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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).
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
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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
15. Assay Analysis
