Colony Transformation

This is a theoretical protocol that has not been tested!

This protocol and be used for the rapid preparation of chemically competent E. coli cells from colonies on a plate for transformation of a high copy number plasmid. The efficiency of this protocol has been reported as 5 x 103 to 5 x 104 colonies per microgram of plasmid. This is 2-200 fold less than for normal chemically competent cells. Thus, it should only be used for an existing plasmid (not a new ligation or cloning product where an even lower efficiency is expected).

Materials

  • 50 mM sterilized CaCl solution
  • LB-antibiotic plate
  • Ice bucket
  • 42C water bath

Procedure

  1. Add 250 l of CaCl2 for each transformation to a test tube. Place on ice.
  2. Transfer one (or more) large colonies from the agar plate to each test tube using an inoculating loop.
    • You are aiming for ~10-25 million cells
    • ALERT! Be careful to not transfer any agar
  3. Resuspend the cells in each test tube by pipetting up and down with a P1000.
  4. Add plasmid to each tube and incubate for 15 minutes on ice.
  5. Heat shock cells by moving tubes to 42C for 30-90 seconds.
    • Time of heat shock depends on thermal transfer properties of the test tube.
  6. Immediately move tubes back to ice for at least 1 minute.
  7. Add 250 l of LB to each tube.
  8. Incubate for 5-30 minutes shaking at 37C if needed for antibiotic marker.
  9. Plate 100 l from each tube on prewarmed LB plates.
    • Plate 40 l of a 104 dilution of the LB culture on a plate without antibiotic in order to estimate the transformation efficiency. You should get ~80-200 cells depending on the size of your colony.

Animated protocol:

References

 Barrick Lab  >  ProtocolList  >  ProtocolsColonyTransformation

Topic revision: r1 - 18 Feb 2016 - 22:29:09 - Main.JeffreyBarrick
 
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