Cell transfection protocol (calcium phosphate method)

Based Maniatis.

2x BBS
50 mM BES
280 mM NaCl
1.5 mM Na2HP04

calcium chloride
1M CaCl2

Cell transfection protocol (calcium phosphate method)

No need to dish out the cells one day in advance before the actual transfection.

  1. The shape of complex DNA: DNA using 20ug and watered in 188uL water, add 62uL of 1M CaCl2, then add 250uL 2x BBS dropwise to calcium chloride-DNA complexes while gently mixing, let the form of calcium phosphate-DNA complexes for 20 minutes)
  2. For 20 minutes, trypsinize plate of confluent cells, turning them down, remove the media, resuspend in new media.
  3. Take the cells resuspended and put ~ 60% to the 1 mL tube.
  4. Rotate and down the cell pellet. After the 20 minutes are up, remove the media, and resuspend each tube into the calcium phosphate-DNA complex.
  5. Let stand for 15 minutes.
  6. Add dropwise to 9ml plate containing the new media. 16-24 hour incubation.
  7. Wash with PBS and media changes the next day.
  8. Cells prepared 48-60 h after transfection day (ie 24-48 hours after changing the medium.).