Bio-Rad MicroPulser™ Electroporator - Manual (Page 18)

7. Resuspend the cell pellet in 25 ml of sterile, ice-cold 10% glycerol. Transfer to a 30 ml
sterile Oakridge tube. Pellet the cells by centrifugation at 12,000 x g for 15 min at 4 °C;
carefully remove the supernatant..
8. Resuspend the cell pellet in 2 ml of 10% glycerol; the final cell concentration should be
~1 x 1010 cells/ml.
9. Dispense 250 µl aliquots of the electrocompetent cells into sterile 1.5 ml microfuge tubes;
freeze the cells in an isopropanol-dry ice bath, then store at -70 °C. The cells are stable for
several months under these conditions.
6.2 Electroporation
1. Pipette the DNA samples (5 ng - 2 µg in a volume ~ 3 µl) to be electroporated into
sterile 1.5 ml microfuge tubes.
2. Thaw the competent cells at room temperature for several minutes. Add 50 µl of cells to
each DNA sample; gently pipette up and down to mix.
3. Incubate the samples at room temperature for 30 min.
4. Set the MicroPulser to "StA". See Section 4 for operating instructions.
5. Transfer the mixture of cells and DNA to a 0.2 cm electroporation cuvette and tap the
suspension to the bottom of the tube. Place the cuvette in the chamber slide. Push the
slide into the chamber until the cuvette is seated between the contacts in the base of the
chamber.
6. Pulse once.
7. Remove the cuvette from the chamber and immediately add 1 ml of SMMP medium
containing a subinhibitory concentration of antibiotic; gently transfer the cells to a sterile
17 x 100 mm tube using a Pasteur pipette. Incubate 1 hr at 37 °C, shaking at 250 rpm.
8. Check and record the pulse parameters. The time constant should be close to
2.5 milliseconds. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
9. Plate aliquots of the electroporated cells on trypticase soy agar containing selective antibiotic.
Incubate plates for 36–48 hrs at 37 °C.
6.3 Solutions and Reagents For Electroporation
1. B2 medium: 10 g casein hydrolysate, 25 g yeast extract, 5 g glucose, 25 g NaCl, 1 g
K
2
HPO
4
; dissolve in 900 ml water and adjust pH to 7.5; bring volume to 1.0 L. Autoclave
2. SMMP: 55 ml 2X SMM, 40 ml 4X Penassay broth, 5 ml 10% (w/v) bovine albumin;
adjust pH to 7.0; filter sterilize
3. Trypticase soy agar: 40 g trypticase soy agar (Becton Dickinson, Sparks, MD) in 1 L of
water. Autoclave.
4. 2X SMM: 25 ml 0.2 M sodium hydrogen maleate, 40 ml 0.1 N NaOH; adjust the pH to
6.5. Add 5 ml 1 M MgCl
2
, 42.7 g sucrose; dissolve and bring volume to 125 ml. Filter
sterilize.
5. 4X Penassay broth: 17.5 g Antibiotic Medium 3 (Becton Dickinson) dissolved in
250 ml water. Autoclave.
6. 0.2 M sodium hydrogen maleate: 11.6 g maleic anhydride or 13.7 g maleic acid, 4 g
NaOH; dissolve in 500 ml water. Autoclave.
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