Berkeley LBL/Mimi-SchlH
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== '''Construction of ''pET3A-(S)-chlH'':'''== | == '''Construction of ''pET3A-(S)-chlH'':'''== | ||
Revision as of 19:31, 26 October 2007
| Home | Project Description | Methods | Notebook | Results and Discussion | Resources |
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 by DNA Gel Electrophoresis is used to confirm that the length of the PCR fragment is the same as the corresponding gene (~4kb).
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. pET3A:
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.
Miniprep cultures.
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
Gel Extraction is performed to isolate the correct band(~5kb).
8. Ligate S-chlH to plasmid pET3A", yielding plasmid "pET3A-(S)-chlH"
9. Transformation into Novablue cells using KCM Competent Cell Transformation using the following conditions:
Plate onto LB Agar + Carb plate
10. Innoculate single colonies into separate 4mL LB + 4mL Carb culture tubes and let grow overnight in 30°C.
11. Miniprep cultures
12. 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
13. Transformation into BL21 cells via KCM Competent Cell Transformation:
15. Protein Analysis using SDS-PAGE
