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Revision as of 06:29, 4 July 2012

Promoters & Reporters workgroup

Bacterial Transformation
Bacteria:T10

Reporters:

RFP: P1-18F K
GFP: P1-14K A
CFP: P1-6A A
YFP: P2/24E AK
OFP: P2/13N K

Violecein P3/12B T
Vio operon ABDE P3/20H K
Vio operon ABCE P3/20J K
CelEBI w/RRS P3/6H A
CelEBY w/RRS P3/6L H

1) Keep constantly the cells on ice
2) Rehydratation of DNA in 10uL H2O 2) Add 1 uL of DNA in 50 uL of T10
3) Incubate 30 min on ice
4) Heat shock the cells during 30 sec at 42 degree Celsius in a water-bath without shaking
5) Put 2 min on ice
6) Add 500 uL of pre warmed SOC-medium
7) Incubate 1h at 37 degree Celsius at 225 rpm
8) Spin at 5000 rpm during 30 sec
9) Remove 150 uL - 400 uL of supernatant
10) Resuspend the pellet in the 150 uL left
11) Spread on appropriate plates
12) Incubate overnight at 37 degree Celsius