Trouble with CaPO4 transfections

Warren Gallin wgallin at gpu.srv.ualberta.ca
Thu Sep 26 17:10:42 EST 1996


In Article <324ACC8D.2682 at mbn1.biochem.nottingham.ac.uk>, Helge Steen
<mbxhhs at mbn1.biochem.nottingham.ac.uk> wrote:
>Hi,
>
>We've been using the CaPO4 method to transfect mammalian cells (eg NIH
>3T3) successfully for years.
>After a recent lab move we are now having problems because the apparent
>transfection efficiency is gradually decreasing. A recent test using
>X-gal staining revealed that the transfection efficiency is approx. 1%.
>This is less than 1/10th the efficiency we had before.
>
>The precipitate we see is comparable to that we saw before the move and
>neither of our assays (luciferase & beta-gal) have changed.

One thing that seems to be absolutely critical is the pH of the 2XHBS. 
variation by as little as .05 of a pH unit can have a big effect on
efficiency.  If you are using a new pH meeting, that might be a problem.

Operationally, you might want to make up several batches of 2XHBS at
slightly different pH values, test them, and select the one that works the
best as the lab stock.  If you make up a liter of each, that should be
enough for a long time.

Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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