User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/02/05

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Transformation of Cells

Objective

Transformation of BL21(DE3) cells with plasmid DNA (T39C L72C Mb) for ampicilin resistance.

Procedure

  1. Thaw on vial of cells on ice
  2. Add 5-10ng of DNA, in a volume of 1-5uL to the cells and mix by tapping gently. Do not mix cells by pipetting.
  3. Incubate the vial on ice for 30 minutes
  4. Heat shock the cells by incubating the vials for exactly 30 seconds in the 42 degree Celsius water bath. Do not mix or shake.
  5. Remove the vial from the 42 degree Celsius water bath and quickly place on ice.
  6. Add 250uL of pre-warmed SOC medium to the vial. (SOC is a rich medium; use proper sterile technique to avoid contamination.)
  7. Secure the vial in a micro centrifuge rack with tape. Place the rack in a shaking incubator, and shake the vial at 37 degree Celsius for 1 hour at 225 rpm.
  8. Plate volume of cells onto LB plates containing appropriate antibiotic for plasmid selection.
  9. Invert the plates and incubate at 37 degrees Celsius overnight



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