DNAse protocol that I am using in my RNA samples says to inactivate DNAse using phenol-cloroform and shows a complex and prolonged protocol to inactivate. There is an alternative method that I can freeze the samples to -80ºC and thaw, that would be much more simple, but they didn't test this method to verify its efficacy and safety for RNA. Can I inactivate DNAse using this alternative method? Is it safe for RNA integrity and work for DNAse inactivation?
Hi Rocha, you can inactivate the DNase I by the addition of 1 µl of 25 mM EDTA solution to the reaction mixture and heating for for 10 min at 65°C. Best wishes.
Dear Amanda
freeze thawing of proteins from -80oC is a way of denaturing proteins in general; including inactivation of DNASe I:
I agree with you that heat inactivation can cause divalent induced cation cleavage of your RNA
- However, in deference to Sharmin, that heat induced hydrolysis of RNA tends to be more pronounced at higher temp, e.g. 95C for 2 min or 85 C for 5 min rather than say 65-70C for 10 min so the latter as stated by Sharmin will alleviate heat induced cleavage.
- I would also say that in relation to Sharmins point that EDTA mops up divalent cations that act as co factors for many enzymes including DNASe I; In fact addition of EDTA following enzyme treatment was routine in the past.
- However, it has fallen out of favour somewhat because EDTA doesn't just inactivate DNAse but also downstream enzymes like rverse transcriptase for example you might want to use
- If you are really concerned about RNA degradation then there are other DNAses out there that utlilise bead technology in order to mop up the DNAses used to treat RNA: Turbo DNASe by PE/ambion is the best of these and I have used it over many years with NO SIGN of degradation (for 1000's of samples EVER:
- https://www.thermofisher.com/order/catalog/product/AM2238
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