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Team:Cambridge/Protocols
From 2009.igem.org
Producing competent cells
Starting from a single colony on a plate:
- Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight
- Take 10ml of the cultture and innoculate into larger LB volume and grow in shaking incubator until OD600 of 1.5 (4 hours?)
- Put culture on ice for 30 minutes
- Centrifuge at 4000g for 6 minutes
- Remove supernatent and resuspend cells in an equal volume of ice-cold 0.1mM HEPES
- Repeat centrifugation
- Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES
- Repeat centrifugation
- Resuspend cells in ice-cold 10% glycerol (20ml)
- Repeat centrifugation
- Resuspend in ice-cold glycerol (3ml)
- Divide cells into 100ul aliquots and store at -80
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)
