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Team:ITU MOBGAM Turkey/protocol
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Revision as of 15:18, 4 October 2013
Protocol
E. coli Calcium Chloride competent cell protocol
1. Inoculate a single colony into 5mL Lb in 50mL falcon tube. Grow O/N @ 37°C.
2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.
3. Shake @ 37°C for 1.5-3hrs.
Or
1. Inoculate a single colony into 25mL LB in a 250 mL bottle in the morning.
2. Shake @ 37°C for 4-6 hrs.
Then….
1. Put the cells on ice for 10 mins (keep cold form now on).
2. Collect the cells by centrifugation in the big centrifugue for 3 mins @ 6000rpm
3. Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
4. Incubate on ice x 20 mins
5. Centrifuge as in 2
6. Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol
7. Dispense in microtubes (300μL/tube). Freeze in -80°C.
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