Calcium Phosphate Transfection
|Refs.||Graham and Van der Eb (1973) Virology 52:456-467
Wigler et al. (1978) Cell 14 725-731
The critical factor in Ca-PO4 coprecipitation transfections is the pH of the solutions. The pH should be checked with nonbleeding sterile pH sticks or reliable pH meters. Since the solutions are of low ionic strength, often 2 or 3 pH meters must be consulted to correctly determine the pH.
The second critical factor in all transfections is sterility. Care should be taken to ensure that the DNA is not contaminated and that all solutions are autoclaved and filtered.
Prepare the following solutions:
- 2X HEBS (HEPES Buffered Saline)
- 8.0 g NaCl
- 0.37 g KCl
- 0.099 g Na2HPO4 (m.w. 141.96)
- OR 0.125 g Na2HPO4 .2H2O (m.w. 177.96)
- OR 0.188g g Na2HPO4 .7H2O (m.w. 267.96)
- 1.0g Dextrose
- 5.0g HEPES
Weigh out ingredients, bring to 450ml with sterile distilled H2O, adjust pH to: 7.2 for plasmid transfections. (7.05 for genomic DNA transfections)
Bring to 500ml with sterile distilled H2O.
- 2.5M CaCl2 in 10mM HEPES
- 0.238 g HEPES (free acid; m.w. 238.3
- OR 0.260 g HEPES (sodium salt; mw 260.3)
- 27.75 g CaCl2
Weigh out ingredients, bring to 90ml with sterile distilled H2O, adjust pH to 7.2. Fill to 10ml with sterile distilled H2O.
- 5.0 ml 1M Tris-HCL pH 7.5
- 2.0 ml 250 mM EDTA pH 8.0
Add 450 ml sterile distilled H2O, adjust pH to:
- 7.3 for plasmid transfections
- 7.15 for genomic DNA transfections
bring to 500ml with sterile distilled H2O
THE FOLLOWING PROTOCOL IS TO TRANFECT ~ 106 CELLS IN 10 ml MEDIA IN A 10 cm DISH
- One day prior to transfection, the cells should be plated such that they are logarithmically growing on the day of transfection (i.e. 50-60% confluent at the time of transfection). The exact amount of cells will vary according to what you are doing. For example, for virus-vector preps where the medium is harvested you might want to plate more cells to increase titer. (For 293T for virus-vector preps use 5 X 106 cells/100. They will be 70-75% confluent the next day.)
- Prepare two sets of 15 ml tubes.
Into one set pipet: 0.5ml 2X HEBS
- Into the other set mix:
- DNA (1 to 20 ug in TE) to 450ul of TE
- 2.5M Ca Cl2 in 10 mM HEPES pH7.2 = 50ul.
Most investigators find that it saves a lot of time and DNA if this reaction is initially performed without DNA to check that the pH of the solutions will allow the formation of a precipitate.
- Mix DNA and CaCl2 by drawing up and down in a 1 ml pipet and add dropwise to the 2XHEBS (NOT THE OTHER WAY AROUND). DO THIS WHILE BLOWING BUBBLES. GET SOMEONE TO SHOW YOU HOW. A fine opalescent precipitate should appear within 10 minutes.
- Let the mixture stand at room temp for 30 min.
- Add the DNA CaPO4 coprecipitate dropwise to the surface of the media containing the cells. Swirl the plate gently to mix.
- After a suitable length of time (overnight for Calcium tolerant cells, 4-6 hours for Calcium intolerant cells), remove the CaPO4 containing medium. Replace with normal medium.
Transient assays for gene expression in transfected cells are performed 48-72hrs post transfection.
Some cells are Calcium tolerant and can live in the presence of Ca overnight, while other cells are Calcium intolerant and should be exposed to the precipitate for no longer than 4-6 hours.
|Calcium tolerant Cells||Calcium Intolerant Cells|