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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Problems doing Northern Blot with small probes (oligo, riboprobe, PCR and random primed)
Posted by: scooperman (IP Hidden, New member, 2)
Date: February 11, 2008 04:40PM
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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