Team:Stockholm/2 October 2010
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==Andreas== | ==Andreas== | ||
+ | ===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX=== | ||
+ | ''Continued from 30/8 transformations'' | ||
+ | ====Colony PCR==== | ||
+ | #<u>BL21</u> pEX.nLMWP⋅SOD⋅His: A & B † | ||
+ | #<u>BL21</u> 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==== | ||
+ | |||
+ | [[image:ColPCR_pEX.CPP_SYoperon_2oct.png|200px|thumb|right|'''Colony PCR gel verification of BL21 pEX.nCPP*SOD*His constructs and Top10 pEX.nCPP*SOD*His.RBS.yCCS operon constructs.'''<br />4 μl λ; 4 μl sample.<br />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'''<br /> | ||
+ | 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 | ||
+ | | rowspan="7" | [[Image:Place_for_picture.jpg|100px|thumb|left|]] | ||
+ | ! well | ||
+ | ! sample | ||
+ | | rowspan="7" | [[Image:Place_for_picture.jpg|100px|thumb|left|]] | ||
+ | |- | ||
+ | | 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 | ||
+ | |} | ||
+ | |||
+ | {{Stockholm/Footer}} |
Latest revision as of 11:04, 26 October 2010
Contents |
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
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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