Primers going bad - what's the best storage/usage conditions? (Dec/13/2006 )
We typically store our master stocks of primers at -80, and make up 10 mM (or micromolar, I forget which, but whichever one "makes sense") working stocks that we keep at -20. Twice in the past six months I have had a primer fail, and when I tried new aliquots, they worked.
I am curious what might make a primer go bad. In lab, we're debating whether we should store them at 4 degrees to avoid freeze-thawing, or -20 or -80... and we are debating the advantages of TE vs DI water for making the aliquots.
Do you have any tips for how to keep primer stocks happy?
Most of the time we use them up quickly enough that there isn't a noticeable problem, but even a problem every few months is too much.
if u r storing ur primers in -20 and they are degenerating, then someone could have introduced contamination or the sol. used to dilute the primers could have been contaminated.
We normally store our stocks in -20 and make fresh dilutions everytime we want to use them.
They havent gone bad.
-20 is ok for the master stock and the working dilutions. The stock should be in 0.1XTE and the working solution in ddH2O, because the excess of salts could affect the results (specially for the QPCR). Always thaw in ice and if their is several persons using the same primer set will be a good practice that each one has their own working solution.
I am in agreement with Scolix, something has contaminated your primer stocks... hopefully only at the working stock level...and not a contamination at the level of the master stock.
I keep my master stocks in TE at minus 20. While my working stocks at suspended in sd water. (so I don't have to deal with the hassle of EDTA inhibition in my PCR reactions.)
There is some degredation of working stock primers... usually corelated with how often it is used. However as my working stocks are rather small and thus remade often (if the primer is in heavy use). I have not actually experienced any out right reaction failures, though there have been occassion where the yields have decreased a little.
Finally: Master Stock Primers only have the best, when ever they are opened, they meet only new tips from an unopen tip box. And worked with a special primer pipette set, kept physically away from the main lab proper.
I normally rehydrate primers in freshly made DEPC water and store my freezer stocks, and working stocks at -20. Working stocks are 10uM in aliquots of 10ul.
After multiple uses the working stocks are fine.