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
• 42°C water bath

Procedure

1. Add 250 µl 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
   • 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 42°C 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 µl of LB to each tube.
8. Incubate for 5-30 minutes shaking at 37°C if needed for antibiotic marker.
9. Plate 100 µl from each tube on prewarmed LB plates.
   • Plate 40 µl 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:

• Animated Protocol – Cell Systems Initiative (UW Bioengineering)

References

• DNA Lab Protocols