Team:Cambridge/Protocols/Transformation of E.coli by Electroporation

Transformation of E.coli by Electroporation

This protocol describes how to transform cells prepared for electroporation using concentrated DNA and an electroporation cuvette.

Theory

Although imaging data was not available at the time of Weaver and Chizmadzhev's review of the "Theory of Electroporation" they discuss detailed indirect evidence and mathematical models which strongly suggest that electroporation causes aqueous pores to form in lipid membranes and that molecules can enter by electric drift. A charged molecule such as DNA is thus very likely to be affected by the electric field applied to cells in electroporation. It might not be fully understood, but its tried and tested all the same.

J. C. Weaver and Y. A. Chizmadzhev."Theory of electroporation: A review " Biochemistry and Bioenergetics. 41. (1996) 135-160

Practice

Preparation: In addition to competent cells and DNA, you will require some SOC media, prewarmed to 37^oC

• Step 1 Prepare Competent Cells as per the Competent Cells for Electroporation Protocol
• Step 2 Dilute your superconcentrated DNA if necessary (Ingenio Electroporation kits recommend the DNA displays an absorbance at 280nm of 1.8 - 2.0, another source suggests 1-50ng per ul is a suitable concentration. (http://userpages.umbc.edu/~jwolf/m7.htm)

Just one ul of DNA is all that is required

• Step 3 Aliquot a small amount, around 1.5ml of SOC medium and keep it to hand, this must be added soon after the electroporation Step X to aid recovery. Ideally the SOC is prewarmed to 37 ^{o<sup/>C, but room temperature SOC can work fine.}
• Step 4

Safety

The safety implication of the procedure.