50 mM BES
280 mM NaCl
1.5 mM Na2HP04
Cell transfection protocol (calcium phosphate method)
No need to dish out the cells one day in advance before the actual transfection.
- 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)
- For 20 minutes, trypsinize plate of confluent cells, turning them down, remove the media, resuspend in new media.
- Take the cells resuspended and put ~ 60% to the 1 mL tube.
- 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.
- Let stand for 15 minutes.
- Add dropwise to 9ml plate containing the new media. 16-24 hour incubation.
- Wash with PBS and media changes the next day.
- Cells prepared 48-60 h after transfection day (ie 24-48 hours after changing the medium.).