Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 77)

2. Add plasmid DNA(s) to the electroporation cuvettes. The amount of DNA required per sample is
dependent on the cell type. Start with a concentration of 10–50 µg/ml.
3. Add cells to the cuvette and tap the side of the cuvette to mix. See Table 8.1 for suggestions on
cell concentrations and volumes to use.
4. Place the cuvette in the ShockPod. Push down the lid to close. Pulse once.
5. Immediately after the pulse, transfer the cells to a plate using ~0.5 ml of media and a plugged
Pasture pipette.
6. Rock the plates gently to assure even distribution of the cells over the surface of the plate.
Incubate the plates at 37°C in a humidified atmosphere.
7. Assay transient gene expression 24–48 hrs following electroporation.
Table 8.1 Suggested starting cell concentrations and volumes for
electroporating mammalian cells
Cuvette Cell concentration Cell volume Growth conditions following electroporation
(cm) (cells/ml) (µl)
0.2 1 x 10 6 100 48 well plate with 0.5 ml growth media
0.2 5 x 10 6 200 6 well plate with 2 ml growth media
0.4 2.5 x 10 6 400 6 well plate with 2 ml growth media
8.3 Reagents and Solutions for Electroporations
1. Growth medium with 10% FBS.
2. Opti-MEM (Invitrogen, Carlsbad, CA).
3. Trypsin-EDTA: 0.05% trypsin, 0.53 mM EDTA in PBS (Invitrogen).
4. Phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 9.5 mM sodium phosphate, pH 7.3;
autoclave.
5. Hepes-buffered saline: 10 mM Hepes, pH 7.3, 140 mM NaCl; autoclave.
6. Phosphate-buffered sucrose: 272 mM Sucrose, 10 mM sodium phosphate, pH 7.3; filter sterilize.
7. Hepes-buffered sucrose: 272 mM Sucrose, 10 mM Hepes, pH 7.3; filter sterilize.
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