(Difference between revisions)
Revision as of 14:50, 31 July 2012
- Thaw a vial of competent cells in ice (100 ul for each transformation).
- Aliquot 100 ul of cells into a 1.5-ml tube.
- Pre-warm a selective LB agar plate and a tube with sterile LB at 37degC.
- Pre-heat the water bath at 42degC.
- Add 1-10 ul of ligation (or 1 ul of miniprepped DNA, or 2-5 ul of resuspended DNA from iGEM plates) to 100 ul of cells.
- Parafilm the vial and incubate in ice for 30 min.
- Heat shock for 1 min.
- Put the vial in ice for 1-2 min.
- Add 1 ml of sterile, pre-warmed LB and incubate the vial at 37degC, 220rpm for 1 h.
- Centrifuge the cells at 10000 rpm, 1 min in a microcentrifuge.
- Remove about 900 ul of supernatant and resuspend the pellet in the remaining liquid, thus concentrating the cells.
- Pipet the resuspended pellet on the pre-warmed LB agar plate and spread evenly.
- Incubate at 37degC overnight or until colonies appear.
- When you don't have enough competent cells, their volume can be decreased from 100 ul to 50 ul.
- When in a hurry, incubation times can be decreased: 5-10 min in ice instead of 30 min; 40 min in LB instead of 1 h.
- When the water bath is not available, fill a becker with hot tap water, put a thermometer in the water and adjust the temperature to 42degC with cold/hot water.
- When in a hurry and using Ampicillin plates, the 1-h incubation in LB can be avoided. Cells mix with DNA can be directly spread on a pre-warmed LB agar plate.
- Heat-sensitive plasmids must be incubated at a proper temperature during the 1-h incubation in LB.