Transformation of chemically competent E. coli cells.
- 20 μL frozen chemically competent cells (standard is JM109)
- 1(3) μL high(low) concentration plasmid
- Incubate competent cells on ice until liquid.
- Add plasmid. Quickly but gently mix and spin.
- 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).
- Heat shock cells 20 s at 42 °C (or special temperature). Quickly return to ice. Incubate on ice an additional 2 min.
- Add 500 μL LB broth.
- Incubate in shaker 40 min 37 °C (or special temperature).
- 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.)
- Incubate overnight 37 °C (or special temperature).
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Sean P Corum *SC 15:40, 23 June 2012 (EDT):
or instead, discuss this protocol.