I'm trying to synthetize a 500 pb of dsRNA. I get my band in a mix with DNA, and I apply a DNase treatment to eliminate this (PROMEGA RQ1 DNase): 15 minutes at 37ºC and the same concentrations of buffer and enzym PROMEGA recomends.
My problem is, each time I do this, my dsRNA is cuted in ~90 pb fragments. I don't know why is happening this.
The only remarkable difference with my sequence is a really high enrichment in AT (except some more "stable" areas which are, by chance, around 90 pb) respect others iRNAs (which can be obtained through this process without any problem).
Can exist some kind of RNase activity in the DNase I???? Or is some other explanation for this selective cutting of my band?
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problem with obtaining iRNA: AT rich sequences???
Started by Sunshadow, May 06 2011 08:17 AM
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chromatin
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Posted 13 May 2011 - 09:47 AM
here is my guess based on what I understood from what you had written:
i think you have rnase contamination. during 37C incubation it's possible that AT rich segment of dsRNA is melted. Single stranded RNA molecules are degraded by RNase contamination. On the other hand, RNAse cannot degrade dsRNA and what you see is ~90bp band. Of course there are dsRNA specific RNasese but it's unlikely to contaminate your samples with it.
i think you have rnase contamination. during 37C incubation it's possible that AT rich segment of dsRNA is melted. Single stranded RNA molecules are degraded by RNase contamination. On the other hand, RNAse cannot degrade dsRNA and what you see is ~90bp band. Of course there are dsRNA specific RNasese but it's unlikely to contaminate your samples with it.
