Team:Birkbeck/Chemical Competent E. coli Cell Protocol

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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 4^oC 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 -80^oC or use immediately.

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