I am using DTT for a qPCR assay I am running. Warned by a few senior lab technicians, I made my one working stocks solution at 50mM in ddH20 and aliquoted into a mess of small tubes using a multichannel pippetman. I then placed opaque boxes of the tubes @-20. When I went back and glanced at the tubes a few days later I noticed that some tubes looked frozen through, while a haphazard bunch of about 10% of the tubes didn't appear to be frozen. Asking around, I have been told that others have observed such things with PCR buffers and other solutions and not to worry about it. However, nobody seems to know quite why. The best we can come up with is that variations in the amount the solution that roll down the side of the tube as they are pippeted might change the local concentrations slightly and change the kinetics of freezing. Any suggestions?
One colleagues suggested not using any tubes which were not frozen.
On a related note, how much does freeze/thawing DTT cause it to degrade? I have a tube of DTT which I have froze-thaw about 5 times over the past few days. I have been getting some inconsistencies across runs and am wondering whether it might be due to degradation in the DTT. Unless you guys tell me this is a silly idea, I intend to test the hypothesis in a few days when I get back in the lab.
Edited by dtae, 14 August 2010 - 10:49 AM.