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ELECTROPORATION OF COS-7 CELLS

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ELECTROPORATION OF COS-7 CELLS

1. Harvest one large (15cm) dish of confluent cells in trypsin and split onto two large dishes. Allow to grow overnight.

2. Next morning cells should be ca. 70% confluent. Harvest using trypsin and resuspend cells in 400l growth medium (10% FCS, 1% L-gln, 1% P/S in DMEM).

3. Electroporations are carried out at room temperature. Place 5l filter sterile 100g/l sheared salmon sperm DNA into electroporation cuvette. Add 50l (1g/l) of DNA to be transfected. Pipette cell suspension into cuvette and allow to stand for 10 minutes.

4. Electroporate cells. Efficiency of transfection is dependent on the charge delivered to the cells. For cos-7 cells, a charge (Q) of 0.15 - 0.20 coulombs is most efficient at producing a high number of transfected cells in a background of untransfected cells, higher charge values cause excessive cell death. Using a higher charge (eg 0.30 - 0.35 coulomb) can be useful for establishing stable cell lines, where following electroporation, a very high proportion of the few surviving cells will be transfected. Q can be calculated by multiplying voltage (V) by capacitance (F) . Settings on the electroporator to deliver a given charge are shown below:

Q (c)V (Kv)C (F)
0.050.40125
0.100.40250
0.150.30500
0.200.40500
0.250.26960
0.300.31960
0.350.36960

5. Allow cells to sit in cuvette for ten minutes following electroporation.

6. Remove cells from cuvette with a pasteur pipette and place into 10ml medium in a medium dish. Allow cells to recover overnight. Change medium the next morning to remove the many dead cells. Assaying for -galactosidase 24 hours after electroporation shows strong staining after two hours to overnight incubation in X-gal.

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This page is maintained by David Bowtell (bowtell@ariel.ucs.unimelb.edu.au) using HTML Author. Last modified on 10/24/95.