RT-PCR with an old DNaseI? - (Nov/14/2005 )
Hello
to avoid contaminations of the RNA with DNA i want to treat the RNA with DNase I, following by a RT-PCR. The problem is, that the only DNase I we have is about 10 years old and i think it could be contaminated with RNase.
Please tell me about your experiences, should i try it without DNase treatment or is there a way to clean the DNase again??
Thanks
Hello
to avoid contaminations of the RNA with DNA i want to treat the RNA with DNase I, following by a RT-PCR. The problem is, that the only DNase I we have is about 10 years old and i think it could be contaminated with RNase.
Please tell me about your experiences, should i try it without DNase treatment or is there a way to clean the DNase again??
Thanks
Storage condition matters for the old DNaseI. Treat your RNA and any DNA sample (e.g. DNA ladder) with the old DNase and run a gel. (FA gel for RNA) If the result is not good enough, buy new one. I don't think RT PCR without DNase treatment is going well.
Thanks for your help,
I already tried the RT-PCR without DNase and my results are completly correct, also my controls. But for the next time i run that gel as you told me
Hi,
DNA fragments may still exist in your RNA extract since you didn't use the DNaseI. So, I think you should still run a gel to comfirm your result eventhough your amplicon size was correct. If DNA fragments exist, then is it convincing enough to say that the PCR product was amplified from your cDNA and not from your original DNA?
I'm just giving a suggestion, I hope it helps.