On 10 Dec 2001, Ruben Calvino wrote:
> 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)
B-cell or pancreatic beta cell line? We do beta cells.
> 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
I have not had the cells die with Ca2+ phosphate, and actually
they normally do very well. Sadly, transfection by this method
is not very good.
> after adding the precipitated DNA, and the
> medium (RPMI 1640) becomes very cloudy.
This tell me there is something wrong with your solutions,
possibly salt concentrations or pH. The salts might be too
highly concentrated or the pH is really far from what you
actually want.
> 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.
The water is usually 0.1X TE
I use following that works in classical system, HEK293 cells
10 µl 2.5 mM CaCl2
2.5 µg DNA
fill to 100 µl with 0.1x TE
add 100 µl HBS
tap on tube a couple times, add immediately to cells.
Too much sodium phosphate can give cloudy appearance,
and you will see MASSIVE crystals all around your cells.
>
> RESULTS. Cells start to die after 3-4 hours. Completely dead after 16
> hours. Cell culture media very cloudy after just 3-4 hours.
>
I have not had cells die, ever, except when the crystals
nearly dominated the solution. In this case, you may
want to have less sodium phosphate, or check the pH of
the final mix (without the DNA of course). This should
be very close to 7.05.
Cheers,
Dominic
North 59 37' 30"
East 17 48' 10"
