I want to amplify a region of a specific pre mRNA, but the standard method is not working.
I know there's a method to do that with RNase H. this enzyme cleaves at sites of double-strand RNA-DNA hybrid.
so you prepare an oligo that will anneal to the site where you want to cleave.
this paper has a protocol: http://nar.oxfordjournals.org/content/36/21/6728.long
Thanks Gal,i will try that. Is there any method which do not require binding of oligo, as i think that the pre-mRNA is having lots of secondary structures and is not accessible to the primers what i am using for cDNA synthesis and amplification ?
Are you denaturating RNA template before cDNA synthesis?
I do 10min 70celsius with Oligo's before synthesis.
Yes, i am using this step which is recommended by Sigma HSRT kit also. But i am not sure if this step is able to denature the 10 kb long fragnment ?
Tarun - If you just want non-specific fragments, then maybe sonication can work?
I know sonication is used to break genomic DNA to small fragments. However, I do not know how sonication affects RNA.
ok..thanks Gal
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