Template:SBB-Protocols Micro1

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

1. Thaw a 200 uL aliquot of cells on ice
2. Put your ligation mixture on ice
3. Add 50 uL of water to the mixture
4. Add 30 uL of KCM salts to the mixture
5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
6. Let sit on ice for 5 min
7. Heat shock for 2 min at 42
8. Put back on ice for 1 min
9. For ampicillin selection, you can plate immediately, otherwise:
10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
11. Plate on selective antibiotics, let incubate overnight