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Northern Blot Background and Non-Specific Probe Binding

Northern Blot Background and Non-Specific Probe Binding - RNA Techniques Forum

Northern Blot Background and Non-Specific Probe Binding - Post and discuss RNA methods and RNA science related topics. RNA extraction, cDNA synthesis, RNA EMSA, and other RNA protocols.


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Old 01-22-2009, 08:16 PM
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Default Northern Blot Background and Non-Specific Probe Binding



I am relatively new to the Northern Blot technique, and I recently performed a blot with a 553 bp purified probe that is supposed to bind to my 1,746 bp RNA fragment. After I ran 10 ug total RNA out on the agarose gel, I took a picture, and the quality appeared to be good with both the 18S and 28S ribosomal subunits appearing very bright with little background. However, when I hybridized the probe to my N+ membrane at 40 degrees celsius overnight and then developed with film both 1 day and 5 days later, I saw that the probe had hybridized to the ribosomal subunits, and those were the only bands that were present. In addition, the background in each lane was very high, so it was hard to even see the 18S and 28S bands on the film. The cells from which I extracted the RNA express low levels of my transcript, so I'm not sure if that also is a problem when performing Northern blot with these samples. So, how can I eliminate the background on these blots and how can I ensure that my probe will bind specifically to my RNA of interest? Thanks so much!
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background , binding , blot , hybridization , nonspecific , northern , northern blot , probe , rna


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