I used EDTA for DNase I inactivation in my samples, but I read that EDTA is Mg Chelator and cause some problem on quality of q-PCR results, and more to that I understand that it has also downstream enzymes like reverse transcriptase and cause some problem in subsequent result, so I checked that using Hprt primer (It’s exon junction primer and only binds to cDNA) for cDNA that came from RNA treated and not treated with DNase I and the result of q-PCR I mean the CT is almost the same(actually, surprisingly the CT of treated samples was a little lower than non-treated), but the problem is that the genes that I want to check doesn’t have that much expression so I think even a little problem can cause some problems and change the result, so I checked other solutions like using phenol/chloroform and freezing and thawing the samples for inactivation of Enzyme, but I think they also have some problem, even though I’m not sure about phenol chloroform protocol, anyway more to all of these things I read that DNase buffer contains relatively high amount of MgCl2, that might cleave/degrade RNA especially during the inactivation step (75°C for 10 minutes), however EDTA chelates the Mg to protect my RNA.
So with all these things what’s the best things to do for DNase I inactivation?
Thank you,
Ali
I believe after the DNAse I treatment, RNA preps can be purified using RNA purification clomuns. This will eliminate any protein such as DNAse I or any other reagent present in previous reaction and give you the pure RNA in buffer of choice for downstream application.
It is very much a matter of concentration. EDTA does not inactivate DNase I, it just removes the Mg ion so that the DNase I no longer active due to lack of Mg. You need to add more EDTA than the amount of Mg ions present to inactivate. If you subsequently (without purification) tried to use a different enzyme that was also Mg dependent, it would fail to work. If you added an excess of Mg to permit the 2nd enzyme to work, the DNaseI would be active again.
So you really need a purification step if you want to remove the DNaseI.
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