TRANSFORMATION OF E. coli by ELECTROPORATION

Preparation of Electrocompetent bacterial cells:

Start a fresh overnight culture with the E. coli strain of interest. Grow at 37° C with moderate shaking.

Inoculate a flask of LB medium 1:200 with the fresh overnight culture, being sure that the flask is large enough to allow adequate aeration. Grow at 37° C with shaking to an O.D.₆₀₀ of 0.5 to 0.6.

Chill the cells in an ice-water bath 15 minutes and transfer to a pre-chilled centrifuge bottle. Centrifuge 20 minutes at 5000 x g, 2-4° C. Resuspend the pellet in 5ml ice-cold water. It is important to keep the cells cold during the entire procedure.

Add the original culture of ice-cold water. Centrifuge as above. Repeat this wash once. Pour off the supernatant immediately and resuspend pellet by swirling in remaining liquid.

If using the cells immediately, place suspension in a pre-chilled, narrow-bottom, 50ml polypropylene tube and centrifuge 10 minutes at 5000 x g, 2-4° C. Resuspend in a volume of ice-cold water to yield approximately 2 x 10¹¹ cells/ml. Aliquot 40-300m l cells into pre-chilled microcentrifuge tubes.

If freezing the cells for later use, add 40m l of ice-cold 10% glycerol, mix, and centrifuge 10 minutes at 5000 x g, 2-4° C. Estimate pellet volume, add an equal volume of ice-cold 10% glycerol to resuspend cells, and aliquot 40 to 300m l into pre-chilled microcentrifuge tubes. Quick freeze on dry ice and store at -80° C.

Electroporation of bacterial cells:

Set the electroporator to desired voltage. Generally for E. coli, field strengths of 16-19kV/cm are required to obtain maximum transformation efficiency. Each strain of E. coli will have an optimum field strength and this will need to be determined empirically.

Add 1m l plasmid DNA (5pg to 0.5m g) to tubes containing 40-50m l electrocompetent cells. Mix. If using frozen cells, thaw the cells on ice immediately before use. Once thawed, the cells must be used or discarded. Any leftover cells may not be re-frozen for later use.

Transfer cell/DNA mixture to a pre-chilled cuvette, incubate on ice 5 minutes, dry the outside of the cuvette, insert into cuvette holder, and place into the electroporator.

Apply the pulse. Remove cuvette and immediately add 1ml SOC medium (without antibiotics) and transfer to a sterile culture tube with a pasteur pipet. Allow the cells to recover by incubating 30-60 minutes with moderate shaking at 37° C.

Plate varius aliquots on LB plates containing the appropriate selection chemical

E. coli Strain	Field Strength (kV/cm)	Transformation efficiency (transformants/m g DNA
C600	19	2 x 10^9
K12	17	3.5 x 10^9
DH5a	17	3 x 10^9
DH10B	16.6	4 x 10^9
Electroporation was performed using the Electroporator 2510 (Eppendorf), 1mm cuvettes, 40m l of cells, and 10pg of pUC19.
Source: Application note: Eppendorf Electroporator 2510. 1-800-421-9988

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