Corum:Transformation

Overview

Transformation of chemically competent E. coli cells.

Materials

• 20 μL frozen chemically competent cells (standard is JM109)
• 1(3) μL high(low) concentration plasmid

Procedure

1. Incubate competent cells on ice until liquid.
2. Add plasmid. Quickly but gently mix and spin.
3. Incubate on ice 10 min. In the meantime, heat water bath to 42 °C. (42 °C is standard. Some special strains may require a different temperature. For example, transformation into KL740 is done at 29 °C).
4. Heat shock cells 20 s at 42 °C (or special temperature). Quickly return to ice. Incubate on ice an additional 2 min.
5. Add 500 μL LB broth.
6. Incubate in shaker 40 min 37 °C (or special temperature).
7. Plate 30 μL cells onto an LB + antibiotic plate with a sterile pasture pipette. (Note, this is for high concentration plasmids. For low concentration plasmids, pellet cells by centrifugation, remove 500 μL supernatant, and re-suspend cells in the remaining 20 μL. Plate this 20 μL onto the LB plate.)
8. Incubate overnight 37 °C (or special temperature).

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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Contact

• Sean P Corum *SC 15:40, 23 June 2012 (EDT):

or instead, discuss this protocol.