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

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

Problems doing Northern Blot with small probes (oligo, riboprobe,PCR and random primed) - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 02-11-2008, 10:45 PM
Sharon
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Default Problems doing Northern Blot with small probes (oligo, riboprobe,PCR and random primed)



Dear All,

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. I am using radiolabeled probes. I have
tried kinased oligo probes, Starfire probes (IDT), riboprobes, PCR'd
probes and random primed probes. I wonder if the problem is that my
message is rare and I don't get enough signal - maybe the random
primed larger probe concatamerizes and increases signal that way, but
I just don't know. I am using Expresshyb (clontech) or Ultrahyb
(ambion) with no difference (no signal). When I decrease
hybridization or washing temperature/stringency I still haven't
gotten a signal (down to 37C with high stringency (2x SSC) wash
buffer only. I can't imagine what I'm doing wrong (I expect it's
something silly and obvious). I would appreciate any advice,
questions, etc.

Thanks in advance,
Sharon
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Old 02-12-2008, 08:44 AM
Peter Ellis
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Default Problems doing Northern Blot with small probes (oligo, riboprobe,PCR and random primed)

Sharon wrote:

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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blot , northern , oligo , pcr , primed , probes , problems , random , riboprobe , small


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