Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 59)

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6. Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 minutes at
4°C; carefully pour off and discard the supernatant.
7. Resuspend the pellet in ~20 ml of ice-cold 10% glycerol. Transfer to a 30 ml sterile Oakridge tube.
Centrifuge at 4000 x g for 15 minutes at 4°C; carefully pour off and discard the supernatant.
8. Resuspend the cell pellet in a final volume of 1–2 ml of ice-cold 10% glycerol. The cell
concentration should be about 1–3 x 10
10 cells/ml.
9. This suspension may be frozen in aliquots on dry ice and stored at -70°C. The cells are stable for
at least 6 months under these conditions.
6.1.2 Electroporation
1. Thaw the cells on ice. For each sample to be electroporated: place a 1.5 ml microfuge tube on
ice, place either a 0.1 or 0.2 cm electroporation cuvette on ice, and place a 17 x 100 mm tube
with 1 ml of SOC at room temperature.
2. To a cold, 1.5 ml polypropylene microfuge tube, add 20 µl of cell suspension if electroporating in 0.1 cm
cuvettes, or 20–40 µl of cell suspension if electroporating in 0.2 cm cuvettes. Add 1 to 2 µl of DNA
(DNA should be in a low ionic strength buffer such as water or TE). Mix well and incubate on ice for
~1 minute. (Note: it is best to mix the plasmids and cells in a microfuge tube since the narrow gap of
the cuvettes prevents uniform mixing.)
3. From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the Bacterial
Protocol screen (press 4, then Enter twice). When using the 0.1 cm cuvettes, press Enter to open
E. coli, 1mm cuvette Protocol Detail screen. When using the 0.2 cm cuvettes, press 2 then Enter, or
3 then Enter, to select the Protocol Detail screens for E. coli to pulse at 2.5 or 3.0 kV, respectively.
See Section 3.4 for operating instructions.
4. Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to
the bottom. Place the cuvette in the ShockPod. Push the chamber lid down to close.
5. Pulse once.
6. Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to the cuvette.
Quickly but gently resuspend the cells with a Pasteur pipette. (The period between applying the
pulse and transferring the cells to outgrowth medium is crucial for recovering E. coli transformants
(Dower et al., 1988). Delaying this transfer by even 1 minute causes a 3-fold drop in transformation.
This decline continues to a 20-fold drop by 10 minutes.)
7. Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37°C for 1 hour,
shaking at 225 rpm.
8. Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The
field strength can be calculated as actual volts (kV) / cuvette gap (cm).
9. Plate on LB plates with antibiotic.
6.1.3 Solutions and Reagents
1. L-Broth: 10 g Tryptone peptone, 5 g Yeast extract, 5 g NaCl; dissolve in 1.0 L water.
Autoclave.
2. LB agar plates with selective antibiotic: prepare L broth as above, adding 15 g of agar per
liter. Autoclave. Cool to 55–60°C and add antibiotic. Pour 12–15 ml per 100 mm plate.
3. 10% (v/v) Glycerol: 12.6 g glycerol (density = 1.26 g/cc) in 90 ml of water. Autoclave or filter
sterilize.
4. TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTA.