Have a 10cm plate of ES cells prepared for electroporation. Ideally you want the plate 70-80% confluent with small to medium size colonies that are smooth and round. One confluent 10cm plate will be enough cells to plate onto 10-12 feeder plates (10 cm).
Prior to electroporating you will need to prepare:
1. Feed cells 2-3 hours before trypsinizing and electroporation.
2. Trypsinize the cells. a. wash cells with 3-5 mls of PBS
b. add 2 mls of trypsin (.05%) and incubate 5 min. at 37C
c. add 5-8 mls of media and titrate
3. Pellet the cells 5-min at 1000 rpms. Discard supernatant.
4. Resuspend in ~0.7 mls of ES media so total volume is ~0.8mls
5. Transfer cell suspension to electroporation cuvette.
6. Add 30ul of DNA to the suspension (30 ug of DNA)
7. Using Biorad electroporator:
a. Connect the chamber directly to pulsar
b. Connect capacitance extender to 2nd readout box
c. Set capacitance box to EXT
d. Set gene pulsar box to 250uF
e. Push the voltage button. Set voltage to 0.32V
f. Slide chamber until latches
g. Push the two red buttons until you hear a beep
h. Check time constant. The range should be 2.8-3.8
8. Let cells sit for 10 min. at room temp
9. Transfer cells to 15ml tube, wash cuvette with ES media
10. Resuspend cells with 10-12 mls of media (depending on number of plates)
11. Plate ES cells onto the feeder plates (which should already have had Feeder media removed and 7ml ES media added – so 8ml total after adding ES cells)
NOTE: Do not add drugs at this time. Feed cells 24 hours after electroporation with appropriate drugs.(i.e. G418, TK, Puro, Hyg, HAT) The plates will be ready to pick 8-10 days later, depending on your cell line, drugs, and confluency.
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