4 replies to this topic

[johnk]

Is it okay to just heat inactivate these nucleases without adding EDTA? I treated my RNA with DNase I and used it to synthesize cDNA which I treated with RNase. In both cases I only heat inactivated (65degrees for 15 mins) the nucleases since I didnt want EDTA to affect my polymerase activity. Should that be okay?

[phage434]

Heat will inactivate DNAse, but not RNAse. Since you'll be working with DNA after your RT reaction, you probably don't care. I would add EDTA in small amounts prior to heat killing -- enough to chelate the Mg++ ions in your buffer. You'll dilute out the EDTA in your PCR reaction, and can (if necessary) add extra Mg++ to the mix.

[tfitzwater]

RNAse A is typically prepared by boiling the stock for 10 minutes to eliminate the contaminating DNAse activity without affecting the RNAse. Heating it to 65C will not affect RNAses.

[johnk]

At what final concentration should the EDTA be added to inactivate DNase and RNase?

[phage434]

The required amounts depends on the magnesium ion concentration (possibly some other divalent species such as calcium, manganese). You need enough to chelate these ions, which are required co-factors for DNAse.