Frozen Competent Cells for CaCl2

Dominik Haudenschild 7-28-93 at CBRC

1) Inoculate 500 ml LB with 1/500 volume o/n culture

2) Grow at 37'C until A600=1.0 (usually 2.5 to 3 hours)

3) Chill Culture on Ice for 10 minutes

4) Centrifuge cells 10 minutes at 8000g

5) Resuspend (by repeated pipetting) in 50 ml Cold 0.1M CaCl2

6) Incubate on ice for 20 minutes

7) Centrifuge cells 10 minutes at 8000g

8) Resuspend cells in 5 ml 0.1M CaCl2

9) Incubate on Ice for 2 to 24 hours

10) Add 1/10 volume of Glycerol, freeze at -70'C in 50ul aliquots