Team:RHIT/Protocols/HeatShockTransformation

From 2014.igem.org

Heat Shock Transformation

  1. Take competent cells from the -80°C freezer.
    • Use DH5α cells in most cases

  2. Turn on water bath to 42°C.

  3. Put competent cells in a 1.5 mL tube. For transforming a DNA construct, use 50 µL of competent cells; for transforming a ligation, use 100 µL of competent cells. You may need more or less, depending on how competent the cells are.

  4. Keep tubes on ice.

  5. Add 1 µg 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 42°C for 45 seconds.

  7. Put tube(s) back on ice for 2 min. to reduce damage to the E. coli cells.

  8. Add 1 mL of LB (with no antibiotic added). Incubate tubes for half an hour at 37°C. If you are trying to grow DNA for ligation, which is more sensitive, leave the tubes for 1 hour.

  9. Spread about 100 µL of the resulting culture on LB plates (with appropriate antibiotic added - usually Ampicillin or Kanamycin- for selection. Grow overnight.

  10. Pick colonies about 12-16 hours later.

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