PCR/dot blot - removing mineral oil
camdna at ubvmsb.cc.buffalo.edu
Tue Apr 27 16:11:00 EST 1993
In article <1rk138INNhp5 at newsstand.cit.cornell.edu>, Adam Law <al16 at cornell.edu> writes...
>I am having problems removing mineral oil from PCR products prior to
>application to a dot blot apparatus.
>My PCR reactions are in 96 well microtiter dishes and so I am trying to
>keep the number of manipulations I make on the products down to the
>minimum. If I take the sample from underneath the mineral oil and then
>apply it to Gene Screen Plus membrane in a dot blot manifold I have
>blotchy results. When I hybridize the membrane with the appropriate
>probe I have blotchy signal in some wells and none in others which I
>reckon is due to droplets of mineral oil blocking the membrane during the
>dot blot procedure. Is there a simple way of removing the mineral oil
>without multiple extractions with solvents or ethanol precipitation? I
>tried adding chloroform to the polycarbonate microtiter dish and this
>just melted the plastic!
>Please let me know if you have any bright ideas
I never tried doing what you are (ie using a 96-well plate and trying to
obtain a sample for dot blotting), but I have sought easy ways to remove
mineral oil from samples. I came up with this:
Freeze the samples.
The aqueous phase will freeze without freezing the mineral oil (at least
if the mineral oil freezes, it will thaw more quickly than the aqueous
phase). While the aqueous phase is frozen, remove the liquid mineral oil
from the top of the sample. Quick and easy, but not guaranteed to
completely separate the phases. Anyway, it will reduce the chance of
contaminating your blot samples with oil.
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