Transcription of Transformation Protocol Using Heat Shock
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Transformation Protocol Using Heat Shock
Transformation Protocol Using Heat Shock MFT, 11/21/03 1) Take competent cells from 80oC freezer. a. Use DH5 cells in most cases. b. If want to cut at XbaI or other DAM- enzyme site, use SCS110 cells which are deficient in Dam and Dcm methylases. 2) Turn on water bath to 42 C. 3) Put competent cells in a ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells. For transforming a ligation, use 100 ul of competent cells. You may need more or less cells, depending how competent they are. 4) Keep tubes on ice. 5) Add 50 ng of circular DNA into cells. Incubate on ice for 10 min. to thaw competent cells. 6) Put tube(s) with DNA and into water bath at 42 C for 45 seconds.
4) Keep tubes on ice. 5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 min. to thaw competent cells. 6) Put tube(s) with DNA and E.coli into water bath at 42οC 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).
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