Cell Transfection Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Cell transfection protocol (calcium phosphate method)

Based on Maniatis.

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

Calcium chloride
1M CaCl₂

Do not need to plate out cells one day in advance before actual transfection.

1. Form the DNA complex: use 20ug DNA and dilute into 188uL water, add 62uL 1M CaCl₂, then add 250uL 2x BBS dropwise to the calcium chloride-DNA complex while gently mixing, let calcium phosphate-DNA complex form for 20 minutes)
2. During these 20 minutes, trypsinize confluent plate of cells, spin them down, remove media, resuspend in new media.
3. Take those resuspended cells and put ~60% into a 1-mL tube.
4. Spin and pellet down cells. Once 20 minutes is up, remove media, and resuspend each tube into the calcium phosphate-DNA complex.
5. Let stand for 15 minutes.
6. Add dropwise to 9mL of plate containing new media. Incubate 16-24 hours.
7. Wash with PBS & change media the next day.
8. Cells ready 48-60 hours after the day of the transfection (i.e. 24-48 hours after changing the media.).