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Team:Stockholm/2 October 2010
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Andreas
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Continued from 30/8 transformations
Colony PCR
- BL21 pEX.nLMWP⋅SOD⋅His: A & B †
- BL21 pEX.nTra10⋅SOD⋅His: A & B †
- 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
† From "Transformation of BL21, 30/8"
Standard colony PCR settings.
- Elongation time: 2:00
Gel verification
4 μl λ; 4 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
0.8 % agarose, 100 V
Expected bands:
- 744 bp
- 765 bp
- 1523 bp
- 1523 bp
- 1553 bp
- 1553 bp
- 1532 bp
- 1532 bp
Results
Ran gel a little too long too see correct clones of constructs 1 and 2. For the other it looks like many clones have double inserts, probably a result of too high insert:vector ratio during ligation.
Confirmed clones:
- -
- -
- A
- B
- -
- -
- B
- B
These will be saved for later.
Mimmi
Over expression
- Load PhastGel
Gel
| well | sample |  | well | sample | |
|---|---|---|---|---|---|
| 1 | ladder | 1 | ladder | ||
| 2 | SOD 0h | 2 | SOD.his 3h 1:4 | ||
| 3 | SOD 50min | 3 | SOD.his sup | ||
| 4 | SOD 3h 1:4 | 4 | his.SOD 0h | ||
| 5 | SOD sup | 5 | his.SOD 3h 1:4 | ||
| 6 | SOD.his 0h | 6 | his.SOD sup |
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