Problems doing Northern Blot with small probes (oligo, riboprobe, PCR and random primed)

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Tue Feb 12 03:44:26 EST 2008


Sharon wrote:
>
>I am having problems doing a Northern Blot with small probes. I am
>able to do a great Northern Blot with a random primed probe that is
>500 bp or longer. However, when I try to do a blot with a 200 bp or
>shorter probe I get no signal.

What are you using as your probe?  Where do they map within the gene you're 
studying?  Have you tried several different short sequences, or is it one 
particular sequence that's failing?  It could (for example) be that your 
200bp probe is derived from an alternatively spliced exon that's simply not 
expressed in the tissues you're investigating.

Next, are you sure that the larger probe is detecting the right thing?  Are 
there repeated sequences in the other 300bp that might cross-hybridize and 
detect some other transcript?  Is the gene you're studying a singleton, or 
is it part of a larger gene family?

Peter 




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