Marianne and Vicki
Protocol: See Transformation Protocol in "iGEM common protocols". It is written below for easy access.
Water bath/heat block at 42°C
LB agar plates (with appropriate selection)
- Remove competent cells (100uL aliquots) from -80°C and thaw on ice.
- Add 1µL ligation mix to thawed cells and incubate for 30 minutes on ice.
- Heat shock in water bath/heat block for 60 seconds and put back on ice for two minutes.
- Add 400uL LB medium and incubate at 37C for 2 hours.
- Spread plate entire 500µL.
- Incubate overnight (<18hrs) at 37°C.
- Add 10uL ligation mix
- Spread on Chloramphenicol plates
- Used competent cells: DH5α
Table 1. Transformation Mixes = Ligation Mix & competent cells
|Transformation Samples||Ligation mix||Competent cells DH5α|
*Note: Could not add 10uL to 10T because not enough (see previous day)
- Chose samples 5,6,11,12 because H171 showed distinct bands on the gel verification of the PCR products
- Chose samples 2,4,8,10 at random - one from each strain: H49 and H80 (of different primers: one with His6-tag and without His6-tag)
Incubate @ 37°C starting 1340
Took out of incubator at 1541
Plated on Chloramphenicol plates - spread 500uL for all
- Note: Did not fully spread 500uL on 4T (did not have enough for some reason - lacking 0.5uL to 1uL)
Began incubation @ 37°C at 1608
Vicki Ma 04:58, 28 June 2010 (EDT)