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Electroporation Procedure
1. Linearize DNA
Use 25 ug/reaction
Check with mini-gel for complete cutting
2. Feed cells 4 hours prior to electroporation.
3. Trypsinize cells with 2 ml trypsin, add 10 ml complete media, spin down, resuspend with 10 ml PBS, wash lx PBS (spin down) Resuspend in 2 ml PBS, count viable cells.
4. Adjust cells to 2.5xlo6 cells/800 ul with DNA present
8 rns 2X,07 cells/6.4 ml PBS
For example: For 12 reactions
1. Put 3X,07 cells in 8.6 ml PBS
2. Add 1 ml cut DNA (12x@5 ug = 300 ug) = 9.6 ml total volume KEEP ON ICE
5. Add 800 ul cell-DNA mix to cuvette, shock. Keep cells/cuvette sterile by adding cell-DNA mix in hood)
6. When shocking, select a range of capacitances and voltages until the conditions best for that cell line are found. (1 setting/reaction).
7. After shocking, put (sterile) the 800 ul of shocked cells into a 50 ml sterile tube. Allow to stand on ice approximately 30 minutes.
8. Add 24 ml RPMI-1640, 10% FCS. Mix. Plate in a 24 well plate at 1 ml/well. This gives 105 cells/well. Put at 370C.
9. After 24 hrs, record the approximate % of cells that have survived this rather rigorous ordeal.
10. 72 hrs after shock: begin G418 selection using the dose of G418 predetermined for that cell line.
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