1 reply to this topic

[centame]

Hi all,

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?

[Trof]

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.