Electroporation of ES cells
| Main Author: | sprotocols |
|---|---|
| Format: | Article Journal |
| Terbitan: |
, 2014
|
| Online Access: |
https://zenodo.org/record/13636 |
Daftar Isi:
- 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. ### Procedure 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). **post electroporation**: 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).