For my PhD project I have developed a set of microsatellites for pinyon pines, but have been unable to publish due to lack of consistent chemsitry. For a while I thought I was being sloppy, but after washing my pipets and the lab bench and redoing a set of reactions, my results kept getting worse. Eventually, I traced my problem to the flask of water that I use when making reagents and PCR reactions. I took the appropriate measures
of getting a new flask, autoclaving fresh Nanopure water and starting fresh. Everything worked beautifully! Then two weeks went by and I was back to the same miserable non-results. Failed reaction after failed reaction, I kept repeating my work since every 10th reaction or so would still function.
Eventually, I did some field work and collected fresh tissue and extracted the DNA from this tissue. Spectrophotometer readings on each sample are mostly spectacular (DNA concentrations of ~150-250ng/ul, 260/280 of ~1.95, 260/230 of ~1.95). I
tried PCR amplification from this new tissue (full strength and 1/10x dilution) and got the same lame result (no desired products, a few random PCR artifacts). Just to be safe, I repeated the PCR but this time using 1/10x DNA diluted in Tris and a "lazy-mans dilution" to 1/50x. Basically, you set up 10uL reactions, aliquot 9uL master mix to each well, add 1uL template DNA to the first set of reactions, mix and spin down, and add 2uL the
first set of reactions to the second set of reactions - voila, 1/50x dilutions with no wasted DNA sample!! I had a few successes, but it no patterns emerged that could help me to consistent success (Gel 1, below)
So I figure I have primer degradation after just 3 weeks. Since I work with pines, notorious for their endogenous suite of PCR-inhibiting compounds, I wondered the other night while falling asleep if I could simply include polyvinylpyrrolidone (PVP) in my PCR reactions to sequester polyphenols that might be contributing to reaction failure. When I got to lab the next morning, I googled the concept and found a paper on just such a thing (http://nar.oxfordjournals.org/content/27/3/915.abstract). So
I prepared a set of of new 1/10x DNA dilutions, this time using my handy stock of PVP-40 rather than Tris to make the dilution. I repeated the 1/10x and 1/50x lazy mans dilution from Gel 1 and things suddenly worked and made sense (Gel 2). As I have long known and practiced, diluting your DNA can reduce the effects of PCR inhibitors in your sample, but the inclusion of PVP can enhance your reaction even more. In gel 2, the first locus (top comb) is more difficult to amplify, (1/10x DNA, no PVP = no PCR product), but at the highest dilution factor and with the inclusion of PVP I get a success of 3 out of 4 reactions. The lower comb, and the easier to amplify locus also gives increasing success in the same way.
So what did I learn? PCR amplification from pines can be enhanced by the inclusion of about 1% PVP-40 in your PCR reaction. You can simply dilute your DNA in PVP-40 rather than adding one more thing to your PCR reaction mix. Initially my water had gone bad, resulting in degradation of PCR primers in a relatively short amount of time. I immediately checked on our Nanopure filtration system and found evidence of bacterial growth in the filters. Probably we should service our filtration device and in the meantime purchase "molecular grade" water from a reputable vendor. While I absolutely hate purchasing water, I hope that this can serve as a lesson to more than just myself.
Happy PCRing!!
