Is it sufficient to heat inactivate DNases and RNases? - (Jun/21/2010 )
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?
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.
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.
At what final concentration should the EDTA be added to inactivate DNase and RNase?
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.