electroporation.doc 11/12/18 Page 1

TRANSFECTION OF CELLS BY ELECTROPORATION

GENERAL INFORMATION

We use the method of Dower [Dower, W.J., Miller, J.F. and Ragsdale, C.W.: High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16 (1988) 6127-6145]. In most cases, the cells are of E. coli strain MC1061, which is F– and has the chromosomal genotype araD139(ara-leu)7696 lac174galU galK hsr–hsm+strAR. The significant phenotypes are restrictionless but modification-proficient for the EcoK restriction-modification system, and resistance to streptomycin (at least to 100 µg/ml).

Electroporation involves lethal voltages (1250 volts in the protocol below); take proper precautions to avoid shock.

There are many electroporators available on the market. If you already have a high-voltage power supply capable of delivering at least 1250 V at 25 mA (2500 V at 50 mA for 2-mm cuvettes), there’s no need to buy an expensive unit with its own internal power supply. In the document electroporator.doc we describe our own homemade apparatus, which cost less than $500 (when we built it in 1991), and which is designed with safety in mind. We use this device in conjunction with the Safety Stand cuvette holder from BTX (Model 630B), but other cuvette holders can be substituted.

Preparation of frozen electrocompetent cells is described in ElectrocompetentCells.doc.

This document presents a 18-µl procedure in 1-mm cuvettes for small-scale ligation tests, clone constructions, etc. A133-fold scale-upfor large-scale library construction is illustrated in RPLconstruct.DOC, steps 53–57.

The nemesis of successful electroporation is arcing (shorting of the cuvette, accompanied by a very loud and startling “pop”). This is nearly always caused by air bubbles in the cell suspension[1]: it’s therefore crucial to avoid introducing bubbles into the suspension before or during delivery into the cuvette. For small-scale electroporations, the problem of arcing has been largely eliminated by layering the suspension onto a red-colored 20% glycerol underlay solution:

20% glycerol underlay (all components autoclaved and mixed in an autoclaved vessel)

6.9 ml water

2 ml glycerol

1 ml TE pH 8

100 µl 10-µM phenol red, neutralized with NaOH to a rosé color (~pH 7) before autoclaving

The red color not only makes it easier to visualize the overlay process, it also makes it much easier to see the “jump” that indicates successful electroporation (see below).

SMALL-SCALE ELECTROPORATION

1. Set up the electroporator at 1250 V and a time constant of 10 ms (for example, 400 Ω × 25 µF = 10,000 µs = 10 ms in our electroporator design electroporator.doc), along with following supplies:

Rack with a labeled 15-ml tube for each electroporation to be carried out,containing 1.5 ml SOC with 0.2 µg/ml tetracycline[2]

Sterile transfer pipette in each 15-ml tube

Space in ashaker-incubator for strapping the rack in

Ice bucket with:

  • Transfecting DNA in low-conductivity buffer such as TE or EB (10 mM Tris.HCl pH 8.5)
  • A 1-mm cuvette for each electroporation to be carried out, containing 55 µl 20% glycerol underlay (see recipe above)

NZY/Tet plates (containing NZY supplemented with 40 µg/ml tetracycline[3])

A bucket of crushed dry ice if multiple electroporations are to be carried out

2. Remove a tube with a 22-µl aliquot of frozen electrocompetent cells for each electroporation to be carried out (seeElectrocompetentCells.doc); if multiple electroporations are planned, put the tubes in dry ice; immediately do each electroporation as follows

Thaw the tube of electrocompetent between finger tips, being careful not to allow the cells to warm up significantly above 0º.

Pipette 1 µl of the transfecting DNA into the electrocompetent cells and stir with pipette tip; leave on ice 30 sec.

Carefully layer 18 µl of the mixture on top of the red 20% glycerol underlay in the cuvette, being very careful to avoid bubbles.

Watch the contents of the cuvette closely as you shock them: the contents should “jump,” indicating that they’ve experienced a high voltage shock. A loud “pop” indicates arcing, and a completely unsuccessful electroporation (see note above)

Using the transfer pipette, immediately draw up the SOC in the corresponding labeled 15-ml tube, use it to resuspend the zapped cells by vigorously pumping up and down a few times, and transfer the resuspended cells back into the 15-ml tube (don’t worry about the small volume that remains inaccessible in the cuvette).

3. When all the electroporations are complete, shake the 15-ml tubes at 37º for 45 min; this is the gene expression period, allowing the antibiotic resistance gene(s) on the transfecting DNA to be expressed before the cells are challenged with inhibitory concentrations of antibiotic at the final step.

4. Meanwhile, label appropriate dilution tubes (capless 2.2-ml tubes; see serial dilutions in stdpreps.doc) to correspond to the labeled NZY/Tet plates next step. Pipette 450 µl SOC into all but the first dilution tube (for undiluted culture). See the note at next step for appropriate dilutions.

5. When the 45-min incubation step 3 is finished, make appropriate serial 1/10 dilutions[4] of each electroporation culture in the dilution tubes by passing 50 µl. Spread 200-µl portions of all appropriate dilution tubes on the corresponding NZY/Tet plates. Incubate plates overnight at 37º.

[1] More rarely, it is caused by too high conductivity in the electrocompetent cells or the transfecting DNA solution.

[2]Here we’re assuming that the transfecting DNA is one of our phage display vectors, which carry the inducible tetracycline resistance determinant of Tn10. Tetracycline at 0.2 µg/ml is sub-inhibitory, but enough to derepress the tetA gene on the phage genome. If the transfecting DNA does not have the inducible Tn10 tetracycline resistance determinant, leave out antibiotic at this step.

[3] Or other antibiotic as appropriate; see previous footnote.

[4] A dilution series ranging from neat (undiluted) to 10-5 will almost certainly cover the possibilities, even if the transfecting DNA at step 3 is untreated RF at high concentration (say 200 µg/ml). For example, here are the results of a transfection with 1 µl of untreated fUSE55 RF at 72 µg/ml:

Dilution / Colony count
10-1 / TNTC
10-2 / TNTC
10-3 / ~1600
10-4 / 186
10-5 / 13
Transfection efficiency / 2.6 × 108 colonies/µg

The observed transfection efficiency of 2.6 × 108 colonies/µg is a pretty good result for untreated RF. For ligations or other highly processed transfecting DNAs at low concentration, the higher dilutions can be omitted.