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 it's 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 (Dilute your superconcentrated DNA if necessary with autoclaved (sterile) reverse osmosis water, Maryland University suggest 1-50ng per ul is a suitable concentration - http://userpages.umbc.edu/~jwolf/m7.htm) You will also require, electroporation cuvettes (1 per sample, labelled) and a 'Genepulse' or similar device, some SOC media, prewarmed to 37^oC and an ice bucket to keep reagents, except SOC, chilled during the procedure.

• Step 1 Prepare Competent Cells as per the Competent Cells for Electroporation Protocol
• Step 2 Aliquot a small amount, around 1.5ml of SOC medium, into a sterile eppendorf for every two electroporation cuvettes and keep it to hand, this must be added soon after the electroporation (Step 6) to aid recovery. Ideally the SOC is prewarmed to 37 ^oC, but room temperature SOC can work fine.
• Step 3 Tilt the cuvette through 45 ^o and pipette around 1ul of your DNA-water solution onto the inner, lower surface of a labelled electroporation cuvette - aim for the ridge slightly above the electrode and keep the cuvette tilted so that the droplet does not move.
• Step 4 With the cuvette still tilted add Xul of competent cells on top of the drop to promote mixing of the cells with the DNA.
• Step 5 Tapping the cuvette on the benchtop can also be performed to aid mixing and move the cells down between the electrodes in the base of the cuvette, but anything more vigorous, such as vortexing, must not be performed as it is easy to damage the competent cells.
• Step 6 load the cuvette into the Genepulse (or similar) device and activate it as per the manufacturers instructions. This should result in a pulse of time constant around 5.5s. (If the time constant displayed is more like 1-2 seconds, and/or a bright flash, or loud pop is heard shortly after activation, it is likely that a high salt content of the DNA or incorrectly prepared competent cells has shorted the device. In this event this sample will not have successfully transformed, and the transformation should be repeated either by making cells competent for electroporation again, or alternatively, try our heat shock protocol.)
• Step 7 Immediately after a successful electroporation, add around 500ul of warm SOC media to the cuvette and incubate the resealed cuvette at 37 ^oC for 45minutes to one hour for recovery before plating or culturing the bacteria.

Safety

None of the reagents used are particularly harmful, but the Genepulse device administers a very high voltage and therefore the electroporation cuvette should only be used with the device in the manner described by the manufacturer, which will normally involve the use of a plastic loading device which connects the cuvette to the electrodes prior to electroporation. If in doubt, seek advice before connecting the cuvette to the device.

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