| Contributor: |
The Laboratory of J. Michael Bishop at the University of California, San Francisco
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1. Feed the cell culture with fresh medium the day before the electroporation.
2. The next day, pellet the cells by centrifugation at 1,500 X g for 10 min. Remove the supernatant and resuspend the cells in PBS and repellet.
3. Suspend the cell pellet in Electroporation Medium (approximately 18 million cells in 0.5 ml).
4. Suspend the DNA in 0.3 ml of Electroporation Medium and proceed at room temperature.
5. In an electroporation cuvette (0.4 cm electrode gap), mix 0.3 ml of DNA solution and 0.5 ml of cell suspension (final volume must be 0.8 ml).
6. Electroporate at 960 microfarads, 300 V.
7. Leave the cells in the cuvettes at room temperature for 5 min.
8. Transfer the partially-lysed and clumped cells with a 1 ml pipette to 1 ml of medium in a 15 ml screw-cap tube. Incubate for 5 to 10 min at room temperature.
9. Break up the clumped cells by pipetting up and down in a 5 ml pipette containing 4 ml of medium.
10. Add this cell suspension to 10 ml of culture medium in a petri dish (15 ml total volume).
11. Assay for expression 2 days later.
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| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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| Electroporation Medium |
| 10 μl BioBrene Plus per 100 ml
0.2% (w/v) Glucose in PBS
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Glucose
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
BioBrene Plus
Sodium Chloride
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No hints are associated with this bioProtocol
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