Overview
This protocol describes how to transform ES cells using electroporation. Electroporation will probably kill about half the cells and about 2 to 20% of the remaining cells will be successfully transfected.
Protocol
- Start with a 10 cm plate of almost confluent ES cells.
- If trying to get stable integration, linearize the plasmid before electroporation.
- Three hours before electroporation change media in plates
- One hour before electroporation warm media and gather supplies
- Prepare four plates for each transfection by coating with gelatin and adding 5 ml media
- Note: If maintaining as stem cells, these should have irradiated MEFs also
- Trypsinze ES cells with 4 ml trypsin/EDTA per 10 cm plate for 5 minutes at 37ºC
- Add 10 ml ES media, transfer to 15 ml conical and spin 5 min 1000 rpm
- Remove supernatant and resuspend in 10 ml PBS (no Ca++/Mg++)
- Count cells
- Spin 5 min 1000 rpm. Remove supernatant and resuspend at 1e7 cells/ml in PBS
- Aliquot 0.8 ml cell solution into a 0.4 mm cuvette (control) (8e6 cells)
- Aliquot 0.8 ml cell solution with 20 - 25 µg of linearized DNA at 1 µg/ml in 0.4 mm cuvette
- Note: Make sure there are no bubbles in the cuvette
- Incubate RT 5 min
- Electroporate with the following settings:
- 250 V 400 µf
- Resistance at infinity
- Cuvette 4
- Note down the time constant (should be 7 to 8)
- Incubate RT 5 min
- Note: There should be bubbles on top from shock and exploded cells floating on top
- Transfer cuvette contents to 50 ml conical containing 20 ml prewarmed media
- Aliquot 5 µl to each of the 4 gelatin coated plates already containing 5 ml media
- Incubate 24 hours before adding selection agent
- Change media every day
- Harvest at 48 to 72 hours