User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/01/16

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Cell Transformation

The objective of this session was to insert plasmid DNA into cells in order to transform the cells. The protocol from New England Biolabs for transformation (C2527) was followed.

Protocol

  1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes

-Because the obtained cells were at -80 degrees Celsius they were left on ice for longer

  1. A stock solution containing the plasmid DNA was made
    1. 9.6ug of DNA were diluted into 960uL of sterilized water
  2. Add 1-5uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place on ice for 30 minutes. Do not mix
  4. Heat shock at exactly 42 degrees Celsius for exactly 10 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 950uL of room temperature SOC into the mixture.
  7. Place at 37 degrees Celsius for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37 degrees Celsius.
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  10. Spread 50-100uL of each dilution onto a selection plate and incubate overnight at 37degrees Celsius. Alternatively, incubate at 30 degrees celsius for 24-36 hours or at 25 degrees Celsius for 48 hours.



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