Cells are routinely passaged two days prior to electroporating. Cells are ready for electroporating when their density is optimal. Usually one 10-cm plate at approx 80% confluency will provide enough cells for 1-2 electroporations. Our standard electroporation protocol is given below.
1. Gelatinize 10-cm plates, and then add 10 mL medium to each.
2. Place them in a 37°C incubator until they are required.
3. Switch on the electroporation apparatus.
4. Harvest the cells by trypsinization.
5. Resuspend the cell pellet in ice-cold PBS (1 mL for each 10-cm plate).
6. Determine the cell density (hemocytometer), and dilute with PBS to the required density for electroporation. We regularly electroporate at a relatively high cell density: 7 x 106 cells/mL (this number varies between different labs).
7. For each electroporation, mix together 20-40 pg vector DNA (for an approx 10-kb vector; see Notes 8 and 9) and 0.8 mL of the ES cell suspension in an electroporation cuvet (Bio-Rad, cat. no. 165-2088) (see Note 8).
8. Set up the electroporation conditions prior to placing the cuvet into the electroporation chamber. We routinely use 250 V, 500 pF for the Bio-Rad GenePulser (see Note 9).
9. Zap the cuvet, then place it on ice for 20 min to 1 h.
10. Transfer the cells from the cuvet into the prewarmed medium containing dishes. (The contents of one cuvette are routinely seeded into two 10-cm dishes).
11. Change medium daily.
12. If drug selection is required, start this on the second day after electroporation (see Note 10).
13. Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 d) (see Note 10).
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