subtraction hybridisaton using Dynal beads:problem?
ilyas m khan
khan at hollywood.u-net.com
Fri Jul 18 18:39:07 EST 1997
I want to do subtractive hybridisation using dynal beads dTTP(18-25) on
which the cDNA driver has been attached/synthesised. that sounds okay, but
i've just realised that if I use this method at low hybridisation
temperature, 37oC, to subtract a population of mRNA, the mRNA will bind to
the beads which are bound statistically to have empty dTTP (18-25) waiting
to take away my precious subtracted mRNA. Now i know that I could raise
the temperature of the reaction to 65oC, but something tells me that that
will give me limited success (probably because the rest of the mRNA will
also fall off, I need to be sure that no mRNA is on those beads.
I phoned up Dynal, who though kind, were unable to help, although they
did suggest that I could swamp the hybridisation reaction with poly AAA,
nice but what happens when I need to make cDNA in the next step, I can see
all this ploy AAA screwing with my first strand synthesis.
my question is how can you block the poly TTTT tail on the dynal beads
so they can't hybridise to mRNA? The greastest minds in Wales have
wrestled with this quandry, now I leave it to bionet!
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