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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