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Team:Valencia Biocampus/Protocols
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=== '''Heat Shock''' === | === '''Heat Shock''' === | ||
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1) Take competent E.coli cells from –80°C freezer. | 1) Take competent E.coli cells from –80°C freezer. | ||
Revision as of 10:48, 29 August 2012
Protocols
Heat Shock
1) Take competent E.coli cells from –80°C freezer.
2) Turn on water bath to 42°C.
3) Put 100 ul of competent cells in an Eppendorf tube.
4) Keep tubes on ice.
5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.
6) Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.
7) Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
8) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
9) Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.
10) Pick colonies about 12-16 hours later.
