Calcium Phosphate Method for Gene Transfection of Mammalian Cells

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Calcium Phosphate Method for Gene Transfection of Mammalian Cells

Based on Maniatis.

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

Calcium chloride
1M CaCl₂

1. Split cells the day before that will reach a confluency of 10-60% confluent on the day of transfection.
2. 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 by holding the cap of the eppendorf tube and flicking gently with the fourth finger.
3. Let the calcium phosphate-DNA complex form for 20 minutes. Try to be reasonably precise with the 20 minutes.
4. While waiting for the complex to form, remove the old media from the plate of cells split yesterday and add 9.5 ml of new media.
5. After 20 minutes is up, add the calcium phosphate-DNA complex dropwise as evenly as possible throughout the plate of cells, while rocking gently with the other hand.
6. 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.). Time may vary to be shorter than 48 hours or longer than 60 hours, depending on how long it takes protein to express and in which cellular location the protein is at a particular time.