Author: Jane McKeating
Calcium Phosphate transfection (Promega kit):
1. Plate 293-T cells in antibiotic free media to reach 30-60% confluence the
following day. 3x105 cells per well of a 12-well plate pre-coated with polyL-lysine (PLL). For PLL coating please see HCVpp generation protocol.
2. Remove media from wells and replace with 1ml pre-warmed Optimem.
3. Thaw all system components to room temperature. For each transfection,
pipette DNA with water first and then add calcium chloride.
2ug plasmid DNA
2M calcium chloride
5.3ul
Sterile nanopure water to a volume of 43ul
4. Dropwise add 43ul of 2x HBS – bubble air through with Gilson pipette and
vortex gently. Incubate the solution at room temperature for 30 mins.
5. Vortex the DNA solution gently before dropping onto cell monolayer –
incubate with cells for 5hrs. Carefully remove media and replace with 3%
DMEM. Monitor antigen expression 48hrs later.
Seeding for large Calcium phosphate transfection of 293-T cells:
Total cell No
cell density
volume per well
6-well plate
1.5x105
7.5x104/ml
2ml/well
6
5
p100 dish
2x10
2x10 /ml
10ml/well.
Following reagents for different culture dishes sizes – scale accordingly to
surface area
DNA
2M Ca Cl2
Water to a volume of:
2XHBS
p60
6-12ug
37ul
300ul
300ul
p100
10-20ug
62ul
500ul
500ul