Weird Northern again!!!
clarosa at biocomp.unl.edu
Tue Jul 25 11:28:49 EST 2000
"Michael L. Sullivan" wrote:
> How are you preparing the PCR fragments for labeling? Are they straight
> out of a PCR reaction, cleaned up somehow, or have you cloned them into a
> vector and then using fragment prepared from that. I was thinking that
> maybe if you are using dircetly out of the PCR reaction, perhaps you are
> labeling something that is relatively low abbundance, but hybridizes well
> to rRNA. Since there is so much rRNA on your blot, those bands could get
> really hot and mask your real signal. Could this be?
In my opinion PCR generated probes are more problematic than say random
primers off of bands cut from gels for the reasons mentioned above..
Unless you run the PCR generated probe out on a gel and run and
autoradigraph, there is no way to be sure your pCR product is the
correct one , at least size and abscence of other bands.
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