Transfection by Ca-phosphate of a B-cell line

Ruben Calvino ruben.varela-calvino at kcl.ac.uk
Mon Dec 10 10:00:42 EST 2001


Dear all,

I'm just new to this world of transfecting mammalian cells and not
very successful at the moment. I'm trying to transfect an
EBV-transform B cell line (Priess) with just the empty vector pcDNA3.1
before trying with the gene I'm interested in. I just started with the
Ca-phosphate method, because of its apparent simplicity, but the cells
are dying just 3-4 hours after adding the precipitated DNA, and the
medium (RPMI 1640) becomes very cloudy. This is the protocol I follow:

DNA precipitation. DNA (15 ug) in 220 ul of water. Add 250 ul 2xHBSS.
Vortex. Add slowly 31 ul 2M CaCl2. Leave for 30 min. The solution
becomes cloudy after that.

CELL TRANSFECTION. 3x106 cells in 5 ml of RPMI + 10% FCS in a 6-well
plate. I add the 500 ul of DNA and incubate the cells at 37oC.

RESULTS. Cells start to die after 3-4 hours. Completely dead after 16
hours. Cell culture media very cloudy after just 3-4 hours.

Any suggestion? What is going on here? I just tried to spin any
remaining cell alive and resuspended in fresh media but during the
spin all that precipitate also goes to the botton of the tube and so
far it doesn't look to improve cell survival.

Any suggestion welcome

Regards




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