CaPO4 Transfection
Note: this protocal has been optimized for C2C12 cells
1. The PM prior to transfection plate 1 x 106 cells/100 mM plate.
2. On AM of transfection change cell media to DMEM, 10% FCS (10 ml/100 MM plate).
3. Spin down ppt DNA (15mg reporter, 5 mg ref plasmid) and sterilely wash with 70% ETOH. Allow DNA to dry in laminar hood.
4. Resuspend DNA in 50O ml of 2 X CaCl2. Pipette up and down 20-30 x and 10' at 37°C.
5. Add DNA/CaCl2 mixture to 15 ml sterile snap cap polystyrene tube (Falcon).
6. Add 500 ml of 2 X HBS dropwise with 1 ml pipette to Ca /DNA solution with continuous mixing. 30' at RT(ppt appears opalescent)
7. Add 1 ml of Ca/PO4/DNA solution to cells, 37°C, 5% CO2 x 5h.
8. Pipette off media, add 1 ml of 15% glycerol solution for 2-3 minutes (time depends on cell type).
9. Rinse plate 2 X with PBS. Add back growth media (DMEM, 20% FCS), 37°C, 5% CO2 O/N.
10. In AM either differentiate cells or split for further growth.
REAGENTS
2 x CaCl2:250mMCaCl2 2.78 g/100ml Store at -20°C
25mM Hepes (Na salt MW 260.3 .65g/100ml #H-7006 Sigma)
2 x HBS(Hepes):250 mM NaCl 1.46 g/100 ml Store at -20°C
25 mM Hepes .65 g/100 ml
1.5 mM Na2HPO4 21.3 mg/100 ml
pH to 7.14 filter sterlize
15% glycerol/HBS 30 ml 50% (w/v) glycerol
50 ml 2 x HBS (7.12) Sterilize store at 4°C
20 ml H2O
Papers Folder 8/2/90
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