Hello,
We are using DNAses 1of Merck, for the removal of DNA contamination from our purified RNA sample. The DNAses 1 amount to be used according to the Merck protocol is 1 unit but this also degrades our RNA, so we use 0.5 unit of DNAses1. But this does not completely remove the DNA contamination. We are following Revert Aid kit method for DNAses 1 treatment. Thank you.
hi dear
why don't you try to increase your washing steps or increase the DNAse units ?!
or you can perform the extraction / purification steps by older method (using trizole , or DEPEC solution )
look this attached procedure may help you
http://www.molecularinfo.com/MTM/C/C3/C3-5/C3-5-3.html
It is not possible for DNAse to degrade RNA, so this is not the problem. You might have RNAse in your preps or RNAse contamination in your DNAse. Ensure you are using RNAse free plastic ware and use new reagents and order fresh RNAse free DNAse. Ensure you aliquot this our as freeze/thaw cycles will drastically effect its efficiency.
Many DNAses like DNAse I has non specific activity it may cleave ss or ds or even DNA/RNA hybrids and its a known fact. So always choose ds specific DNAse which targets only DNA not RNA. I have read that Shrimp Nuclease is the type which is safe to treat RNA samples. U can search it through your supplying companies... Never look just the name that it is DNAse and hence it will only degrade DNA, no not at all, you need to look about the details of enzyme spectrum u wanna use. We got decrease RNA yield by treating samples with DNAse and then we got this information..
Please have a read of this:
http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/nucleases.html
DNase WILL NOT degrade RNA but can be potentially contaminated with RNAse.
Shrimp DNase shouldn't be used in RNA isolations:
http://www.affymetrix.com/catalog/131318/USB/Shrimp+DNase+Recombinant#1_1
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques