Team:Stockholm/5 October 2010
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Andreas
Transfer of pEX.nCPP⋅SOD⋅His to BL21
Glycerol stocks
- BL21 pEX.nLMWP⋅SOD⋅His
- BL21 pEX.nTra10⋅SOD⋅His
Assembly of His⋅SOD⋅cCPP constructs
Colony PCR
- pSB1C3.His⋅SOD⋅cTra10 A-D
- pSB1C3.His⋅SOD⋅cTAT A-D
- pSB1C3.His⋅SOD⋅cLMWP A-D
Standard colony PCR settings
- Elongation time: 1:30
Gel verification
1 % agarose, 120 V
Expected bands
- 884 bp
- 854 bp
- 863 bp
Results
Varying band sized, with the following clones seeming correct:
- A
- D
- B & C
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Plasmid preps
From 4/10 ON cultures
As I'm still waiting for sequencing results of the operons in pSB1K3, I spun down cells (4,400 x g, 10 min), and saved pellets in -20 °C ON.
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: B