unspecific (was: RNA stability) PCR probes???

Mart Speek smart at KBFI.EE
Wed Nov 24 02:02:54 EST 1999


Susanne wrote:

I am using an internal DIG-labeled PCR fragment of my gene as a probe.
so that shouldn't be the problem. maybe I should go and check for
homologies with the rRNA. I hope you won't go "but DIG-PCR probes never
work"... It should, shouldn't it?
---------------------------------------------------------
$$$ Strictly speaking, in more serious experiments,
I wouldn't use PCR derived probes for hyb.
This is because PCR can always have some side products,
which you label up similarly as the major product.
As a hyb-probe/mixture of probes is normally added in excess,
you could see multiple hyb signals. This is exactly what you
don't want. I am sure most folks dot believe
that this could be the case, however, this is what my colleagues
have FREQUENTLY seen/shown before.

Regarding the probe similarity to rRNAs, it is always a good
idea to check the oligo-matches first. (a five-minute experiment, I call
it!)

Good luck,

Mart (ESTONIA)








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