Latest revision as of 02:58, 20 July 2018

This is a simple minimal protocol for E. coli heat shock transformation that works and has been repeatedly successfully used in an undergraduate classroom.

Protocol

1. Grow streaked cells overnight at 37 C on a plate.
2. Heat a water bath to 42 C.
3. Label a microcentrifuge tube.
4. Chill 500 µl of 0.1 M CaCl₂ in a microcentrifuge tube on ice for 5 min.
5. Scrape up a colony with a wire loop (or equivalent) and transfer to CaCl₂ solution mixing slightly to break up the cluster of cells.
6. Add ≈10 µl of DNA to the cell/CaCl₂ mix and stir gently.
7. Incubate the cells with the DNA in CaCl₂ on ice for 15 min.
   1. It is very important to keep the cells on ice and make the next change in temperature the cells are exposed to as abrupt as possible. Do not take the microcentrifuge tube out and carry it in your hand over to the water bath. Carry the container of ice over to the water bath with the tube in it.
8. Holding only the top edge of the tube cap quickly immerse the tube in 42 C water for 90 s with a rotating stirring motion.
9. Immediately transfer the tube back to the ice.
10. Spread the cells on a plate, usually with selective media containing an antibiotic such as ampicillin, and grow overnight at 37 C.

What is happening?

Alternatives

Notes

Could TRIS increase transformation efficiency? See http://jb.asm.org/content/145/3/1397.full.pdf

What Links Here

• Genetic Transformation ‎ (← links)