Problems with DNAse treatment - (Jan/05/2009 )

Hello, everybody! I'm with a big problem, so I registered in here to see if somebody could help me. First, sorry for my bad English... Well, I'm trying months to do a DNAse I treatment with my trizol extracted RNAs. After the treatment (with addition of EDTA, like the protocol), I reextracted the RNAs and I run an agarose gel. The RNAs are good, but when I do a PCR with the cDNAs synthesized using them, there's no bands in the tester lanes, and appears a band in the negative control lane... I always use a mix to pippete my reactions... So I'm going crazy with this problem! Please, help me to understand this!!!

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Have you tried RT and PCR on RNA that hasn't been DNase I treated?

Is the band that you see in the negative control the correct size?

Also, for good quality DNase I (i.e. DNase which is RNase free because it lacks RNase and not because they've added RNase inhibitor) the extraction step is not necessary. Just heat inactivate it and run your RT.

I treat 80ul of Trizol extracted RNA (about 950ng/ml total nucleic acid, including RNA and contaminating DNA) with 10 units DNase I in a 100ul reaction for 60 min at 37C. Then I heat inactivate at 72C (70C is sufficient but my heat block always reads 72C) for 6 or 7 min. My cDNA always comes out nicely.

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