Primer Degradation - What exactly happens? (Jun/14/2010 )
Using standard PCR protocol with published primers, I had no problems initially. Over time, erratic results, finally no results. All components check out bar the primers. My question is: Primers are DNA, therefore a stable molecule - so what causes "degradation." Is it solely down to inadvertent contamination by nucleases? Is it due to too many freeze-thaws? What actually happens during repeated freeze-thaw? Do the primers simply break down into nucleotides or shorter primers? Do they start to fold back on themselves? Ran a spec analysis from 220 nm to 320 nm with nothing to suggest that the DNA was "degraded" I just find the word "degradation" seems to be a very convenient catch-all term that explains away poor results, yet I do not know what is really happening in the stock. Also, is there a recommended buffer for reconstituting primers. I merely rehydrate using PCR-grade or Molecular Biology-grade water. Oh, and I store at -20, not -80? Anything to worry about over storage temperature?
I only know I once had a problem with degraded primers stored -20 in MB-grade water and since then I'm using 10mM Tris pH 8 (which is actually only little more than buffered water) to reconstitute and dilute my primers and DNA in general. I was told the the alkaline pH should prevent nucleases from degrading the primers, but I'm not sure about the underlaying mechanism.