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Team:Stockholm/4 October 2010
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===Transfer of pEX.nCPP⋅SOD⋅His to BL21=== | ===Transfer of pEX.nCPP⋅SOD⋅His to BL21=== | ||
====Gel verification==== | ====Gel verification==== | ||
| + | [[image:ColPCR_BL21CPP_4oct.png|200px|thumb|right|'''Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.'''<br />4 μl λ; 5 μl sample;<br />λ = O'GeneRuler 1 kb DNA ladder.]] | ||
''Re-run of 2/10 BL21 samples'' | ''Re-run of 2/10 BL21 samples'' | ||
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====Gel verification==== | ====Gel verification==== | ||
| + | [[image:ColPCR_CPP-SH-Ry_operon_4oct.png|200px|thumb|right|'''Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.'''<br />4 μl λ; 5 μl sample;<br />λ = O'GeneRuler 1 kb DNA ladder.]] | ||
0.8 % agarose, 100 V | 0.8 % agarose, 100 V | ||
Revision as of 15:11, 4 October 2010
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Contents |
Andreas
Transfer of pEX.nCPP⋅SOD⋅His to BL21
Gel verification
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.
Re-run of 2/10 BL21 samples
- BL21 pEX.nLMWP⋅SOD⋅His: A & B
- BL21 pEX.nTra10⋅SOD⋅His: A & B
1 % agarose, 120 V
Expected bands:
- 744 bp
- 765 bp
Results
- Both clones verified
- Clone A verified; weak band for clone B
ON cultures
- 3 ml LB, 30 °C
- A: BL21 pEX.nLMWP⋅SOD⋅His
- A: BL21 pEX.nTra10⋅SOD⋅His
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Colony PCR
Picked 2 new colonies of each of the two constructs transformed 30/8:
- 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
- 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
Standard colony PCR settings
- Elongation time: 2:00
Gel verification
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.
0.8 % agarose, 100 V
Expected bands:
- 5. 1553 bp
- 6. 1553 bp
Results
- 5. Relevant band for clone B; too large insert (double?) for clone A.
- 6. Relevant band for clone B; too large insert (double?) for clone A.
ON cultures
- 5 ml LB, 37 °C, 250 rpm
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B
Verification of pSB1x3 plasmids
Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:
- pSB1A3
- pSB1C3
- pSB1K3
- pSB1AC3
- pSB1AK3
