Mammalian Cell Transfection Calcium Phosphate Method
Day 1: The day before transfection, split cells so they will be 50 60% confluent on day of transfection.
It is important that on the day of transfection the cells are thoroughly separated on the dish,--also keep in mind the optimal density that produces a near confluent dish when the cells are to be harvested or split into selective medium.
Day 2: Transfection:
(1) Add 500 ml of 250 mM CaCl2 and DNA to sterile eppendorf tubes, mix by flicking/tapping tubes
The DNA should be purified twice by CsCl gradient and ethanol precipitated to make it sterile. The total amount of DNA should be 20 mg. Use approximately 2 10 mg of plasmid of interest (depending on transfection efficiency and expression level desired). Use empty vector plasmid carrier DNA to make up the difference in total DNA. If co-transfecting with a separate plasmid carrying a selection marker,--use at a 20:1 ratio with plasmid of interest.
(2) In either a 15 ml sterile polystyrene tube or RIA tube mix 500 ml 2XHBS and 20 ml 100XPO4.
(3) Add the DNA/CaCl2 to the HBS/PO4 drop-wise while vortexing
Vortex setting should be low such that gentle mixing occurs. This is done to create as fine a precipitate as possible.
(4) Incubate 20 30 minutes at room temperature
Solution should be cloudy and not chunky. If precipitate too large, cells will not take it up.
(5) Add solution drop-wise to cells and swirl.
One 10 cm plate containing 10 ml of medium is transfected with 1 ml of precipitate solution at a DNA of interest concentration of 2 10 mg/ml; use 0.5 ml for a 60 mm plate containing 5 ml of medium.
The medium will become yellow-orange and turbid when adding the precipitate.
(6) Depending on cell type, wash cells two times with 1xPBS and re-feed with growth medium, 6 8 hours post-transfection or overnight. Look at cells, dont want to see any precipitate on the cells, if so,--wash one more time with medium.
The time is related to the toxic effects of the precipitate being on the cells for too long. The precipitate can be left for up to 16 hours on 3T3 cells.
(7) For transient expression, harvest the cells 48 60 hours after transfection
For assays that involve replicate samples or treatment of transfected cells under multiple conditions or over a time course, it is desirable to avoid dish-to-dish variation by transfecting large monolayers of cells and then trypsinizing the cells after 24 48 hours of incubation and distributing them among several small dishes.
SOLUTIONS: all should be sterilized by filtration
2XHBS: Hepes 10g/l
Bring to pH 7.1 + 0.05
Store in 5 ml aliquots at 20oC
Efficiency can vary with each batch. Test by mixing 0.5 ml 2XHBS with 0.5 ml CaCl2. A fine precipitate should develop that is visible in the microscope.
100XPO4: mix 1:1 70 mM Na2HPO4 and 70 mM NaH2PO4.
Store in 1 ml aliquots at 20oC
250 mM CaCl2 Store in 5 ml aliquots at 20oC
1XPBS: NaCl 8g/l
Na2HPO4 1.15 g/l