Difference between revisions of "Team:Birkbeck/Chemical Competent E. coli Cell Protocol"
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Latest revision as of 17:23, 18 September 2015
Preparing chemically competent cells
Making chemically competent cells
- 1. Grow 5 mL overnight culture in LB broth.
- 2. Sub-culture (1% inoculum) in 25 mL of LB.
- 3. Grow until OD600 = 0.3-0.5 (DH5a, this is around 2 hours).
- 4. Put on ice for 20 mins (can be longer, not an essential step).
- 5. Spin cells down at 4oC at 4,500 rpm for 20 mins.
- 6. Resuspend in 2.5 mL of ice cold TSB.
- 7. Leave on ice for at least 10 mins.
- 8. Snap freeze in 50 uL aliquots to -80oC or use immediately.