difficult Northern

Abelardo Solano via methods%40net.bio.net (by asolano from ochoa.fib.es)
Wed Feb 24 07:27:16 EST 2010


Hello everybody!;
I´m having a very difficult time trying to hybridize a RNA-Dig
labeled probe, 55% GC, to total RNA through Northern blot. I know my
RNA probe has a strong secondary structure; I denature at 100ºC for
10 minutes before hybridization at 65ºC overnight.
All RNA probes I use for other genes (as well as control genes) are
working fine. Actually I improved my Northern conditions using the
protocol by Van Vliet that was published here in 2000, but my problem
still remains for the RNA I want to detect. Translation seems not to
be the problem, since there is no degradation of RNA
and the T7 promoter is in the correct position of the template.
Dot-blot with RNA always gives signal (I guess it is not-specific).
Anyone can give me a good practical formula to calculate the Tm of my
RNA probe (220 bp in length)? And Thanks for any other hints you may
have. I have spent a lot of time with this matter.  
ABe S.
Centro de Investigación Príncipe Felipe
Valencia, Spain.





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