Preparation of Competent Cells for Electroporation

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Purpose

This protocol is used for preparing competent cells to be used in electroporation transformation.

Materials Needed

-LB Broth

-28 C incubator

-37 C incubator

-centrifuge

-microfuge tubes

-ice cold 10% glycerol

-micropipette

-cells to be transformed

Protocol

-Inoculate 5 mL of LB broth with bacterial cells to be transformed and grow overnight at 28 C with shaking

-Transfer 2 mL to 200 microliters of LB broth in a 1 liter flask and grow at 37 C with shaking until the optical density is 0.3-0.5

-Centrifuge the cells at 10,000 rpm for 10 minutes and discard the supernatant

-Resuspend the cells in 35-40 mL of ice cold 10% glycerol and centrifuge at 10,000 rpm for 10 minutes, discard the supernatant

-Resuspend the cells in 18-20 mL of ice cold 10% glycerol and centrifuge at 10,000 rpm for 10 minutes, discard the supernatant

-Resuspend the cells in 350-400 microliters of ice cold 10% glycerol

-Aliquot the cells into small tubes of about 40 microliters each

-The cells can be used right away or frozen in liquid nitrogen and stored for later use

Papers where this or a similar method has been used

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References

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