Revision as of 05:21, 13 November 2012 by
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 1 ml 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.
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