COMPETENT CELLS FOR ELECTROPORATION

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You will need:

- 1 L sterile LB in a Fernbach flask
- ~2 L sterile H2O
- 4 sterile GSA rotor bottles/lids (250 ml)
- 2 sterile SS-34 rotor bottles/caps (50 ml)
- a good part of a whole day

Keep the cells and all materials cold throughout procedure!!

1. innoculate 3 mls LB + antibiotic with a single (fresh O/N) colony
2. innoculate 1 L LB + antibiotic with this 3 ml culture
3. grow cells (37 C/shaking) to 0.5-0.8 OD(600) (cell strain-dependent)
4. chill cells on ice 15-30 min.
5. spin down cells 15 min. (4K rpm) @ 4 C, remove as much SN as possible
6. suspend cells in 1 L ice cold H2O
7. spin down cells as in step 5.
8. suspend cells in 0.5 L ice cold H2O
9. spin down cells as in step 5.
10. suspend cells in 20 mls ice cold sterile 10% glycerol
11. spin down cells as in step 5.
12. suspend cells in 2.5 mls ice cold sterile 10% glycerol
13. quick freeze ~125 ul aliquots on dry ice and store -80 C

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made Feb. 22, 1999; Baker Laboratory

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