I am currently using Ambion DNAse to remove any DNA contamination from my RNA samples. The protocol requires a 37C incubation for 30min and then a 75C incubation for 10min to deactivate the enzyme. The protocol also says to add EDTA to prevent RNA denaturation during the hot incubation. I think the protocol implies that the EDTA should be added after the 37C incubation but i'm not sure. Here's the link to the protocol (https://docs.google.com/viewer?url=http://tools.lifetechnologies.com/content/sfs/manuals/4393898B.pdf ). Does anyone know if the DNase enzyme will still effectively remove the DNA if the EDTA is present during the 37C incubation?
Richard, almost all nucleases require divalent cations for their action. DNaseI, which is usually derived from pancreas, is dependent on Mg2+ (contrast it with the other popular nuclease, MNase, which is dependent on Ca2+). EDTA is a chelator that traps divalent cations (preferably Mg2+), and hence deprives DNaseI of this essential ingredient. This is how EDTA inhibits DNaseI. If you have EDTA during the 37C incubation, you should expect NO DEGRADATION of the contaminating DNA at all! So, it is imperative that you add EDTA ONLY AFTER the 37C incubation. While 75C incubation will effectively kill all DNaseI, do NOT do it without first adding EDTA! Since the reaction contains Mg2+, higher temperature will kick in cation-dependent cleavage of RNA. So, before that temp elevation, you must sequester all Mg2+ ions available! Hope it is clear to you now.
EDTA will also inactivate DNase I in addition to RNases. When the protocol mentions adding EDTA it is specifically for the enzyme denaturation step for the DNase I not the 37 C incubation.
Richard, almost all nucleases require divalent cations for their action. DNaseI, which is usually derived from pancreas, is dependent on Mg2+ (contrast it with the other popular nuclease, MNase, which is dependent on Ca2+). EDTA is a chelator that traps divalent cations (preferably Mg2+), and hence deprives DNaseI of this essential ingredient. This is how EDTA inhibits DNaseI. If you have EDTA during the 37C incubation, you should expect NO DEGRADATION of the contaminating DNA at all! So, it is imperative that you add EDTA ONLY AFTER the 37C incubation. While 75C incubation will effectively kill all DNaseI, do NOT do it without first adding EDTA! Since the reaction contains Mg2+, higher temperature will kick in cation-dependent cleavage of RNA. So, before that temp elevation, you must sequester all Mg2+ ions available! Hope it is clear to you now.
Thank you for explaining the details of the science behind not adding EDTA before the 37C incubation. I knew there had to be a logical reason and now i know what it is :)
Hi,
in this regards there are two querries of mine
1. If I just want to get rid of DNA from a mix which has to be further used for amplification of gDNA , can I first use the DNaseI to degrade the contaminating DNA followed by only heat inactivation (95 for 15 mins).
2.Can DNase I renature at the lower temp ...say i store the treated mix at 4
..thenm??
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