User:Reef Koh Junjie/Notebook/Expression of p23 protein/Entry Base

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Transformation Protocol Using Heat SHock

  • Transformation Protocol Using Heat Shock

1) Take competent E.coli cells from –80oC freezer. a. Use DH5α 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 42οC. 3) Put competent cells in a 1.5 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 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). Incubate tubes for 1 hour at 37 οC. (Can incubate tubes for 30 minutes, unless trying to grow DNA for ligation which is more sensitive. For ligation, leave tubes for 1 hour.) 9) Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added – usually Ampicillin or Kanamycin.) Grow overnight. 10) Pick colonies about 12-16 hours later.