Author: Nagy Lab
Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations.
1. Change medium on ES cells 3-4 hours prior to electroporation
- Gelatinize 10 cm plates, then add 10 ml medium to each.
- Place them in a 37 0C incubator until they are required.
- Switch on the electroporation apparatus.
- Harvest ES cells by trypsinization.
- Resuspend the cell pellet in ice-cold PBS (1 ml for each 10 cm plate).
- Determine the cell density (haemocytometer) and dilute with PBS to the required density for electroporation. We regularly electroporate at a relatively high cell density: 7x106 cells/ml (this number varies between different labs).
- For each electroporation mix together 20-40 microgram (1 µg/µl) DNA (for an approximately 10 kb vector) and 0.8 ml of the ES cell suspension in an electroporation cuvette (BioRad, Cat. No. 165-2088).
- Set up the electroporation conditions prior to placing the cuvette into the electroporation chamber. We routinely use 250 V, 500 microF for the BioRad GenePulser.
- Zap the cuvette, then place it on ice for 20 min to 1 hour.
- Transfer the cells from the cuvette into the prewarmed medium containing dishes. (The contents of one cuvette are routinely seeded into two 10 cm dishes).
12.Change medium daily.
13.If drug selection is required start this on the second day after electroporation.
14.Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 days).