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!!
Hi, I have a stupid question, when you said that you used pvp at 1%. Do I have to prepare a stock solution at 1% or 100% and then the finally conc. into my PCR reaction is 1% ? . PCR final vol 25 ul. by the way sorry for my bad english
ReplyDeleteI typically make a working stock of 10% and use that into your PCR reaction. Since I prefer very small reactions (<10uL), this still allows for a pipettable volume.
ReplyDeleteThank you for answer me, I have a last question i only have pvp Wt: 360,000 I have been searching about pvp but looks like everybody use pvp MW 40,000 .... I don't know if is good idea try with this one. do you have any recommendation? thank a lot again :)
ReplyDeleteI don't know of any specific problems with PVP360. I have used it in the past without issue, and I did so because like you I had only PVP360 on hand. Eventually I purchased PVP40 like everyone else uses. PVP360 will be a longer chain so it seems like it should simply have more polar binding sites than PVP40. I might consider reducing the concentration of PVP360 relative to PVP40 because of this. If pure, PVP360 should have 9X more binding capacity than PVP40. My gut tells me that this won't be a strict stoichiometry and adding PVP360 at 1/9th the usual concentration of PVP40 might not be as effective as a common PVP40 solution. I might try a range of PVP360 concentrations first to see if it does anything for you. To test, I would probably do 4 reactions each with 1%, 2%, 5%, and 10% PVP360 (w/v) and use the concentration which perks up your PCR the best.
ReplyDeleteLet me clarify. These are the final concentrations of PVP meant for DNA dilutions. Dilute your DNA down maybe 50% in the process. Alternatively, have you tried a dilution series PCR? This sometimes works very well. Set up a series of 3 10uL PCR reactions (make the master mix at once). Aliquot 9uL mix to each well. Add 1uL DNA sample to the first reaction, mix spin down. Then take 1uL of the first reaction (the one you just put DNA into) as template DNA for the second reaction. Then a uL the second reaction as template for the third. You now have created 1x, 1/10x, and 1/100x dilutions of your sample without wasting any sample. The volumes will be slightly different, yes, but the concentrations will be the same. Often I find this is the best way to resolve poor PCR performance.
ReplyDelete