CaPO3 Transfection in 293 T cells

 

  1. 24 hours before transfecting, plate out cells at 1.25 x105/well in a 6 well plate (106/100 mm plate). The density should be about 25%. Too low — the cells will die; too high — they will label inefficiently and overgrow the plate before harvesting.
  2. Check pH of buffers (critical). Combine 100X NaPi with 2X HEPES in a 1:50 ratio, so that the final concentration of NaPi and HEPES is 2X. Filter sterilize all buffers in the cell culture hood.
  3. Label sterile eppendorf tubes. For every well to be transfected, resuspend 5mg of CsCl double-banded DNA in 150 ml of CaCl2. (10ug DNA & 300ml of CaCl2/ 100mM plate)
  4. Bubble the CaCl/DNA mixture with a 1ml sterile pipette and add an equal volume of the NaPi/HEPES solution dropwise (SLOWLY). Leave for approximately 30 min.
  5. Gently add the DNA/CaPO3 precipitate to the cells swirl gently to distribute the DNA.
  6. Incubate the cells and DNA for 8-16 hours. Gently remove the media and replace with 2 ml of complete DMEM. Metabolically label cells the following night and IP 36-48hrs after transfection.

Solutions:

100X Sodium phosphate (NaPi)

35 mM Na2HPO4 (dibasic sodium phosphate)

35 mM NaH2PO4 (monobasic sodium phosphate) in ddH20.

2X HEPES buffered saline solution

1% HEPES

1.6% NaCl, pH 7.1 (pH of this solution is critical)

250 mMCaCl2 in TE, pH 7.1. (pH of this solution is critical)

 

 

 

 

 

Nb Alans original protocol:

7.5ug DNA in 300ul CaPo3 and added 2ug of sheared SS DNA when not cotransfecting

Changes the medium prior to addition of the precipitate

Glycerol shock Option:

Remove medium and add 1ml of 15% glycerol in TE for exactly 1 min

Wash with HBSS

Add 3 mls of medium

 

WHEN SCALING UP

There are problems with sterility if pipettemen have to be used to put DNA into 15ml tubes

If there are many samples there may be a problem with DNA degrading after Ca addition - process in batches of less than 10