Tips for tricky PCR

Use mitochondrial DNA to identify ancient samples.When a creature has spent years in the back of a cave, "it basically is nutrients for all the microbes and worms in the environment," says Edward Rubin, director of the US Department of Energy's Joint Genome Institute in Walnut Creek, Calif. With few intact cells remaining, the chances of finding nuclear DNA extraction are slim. However, there are a few thousand copies of the same mitochondria per cell, so aim for mitochondrial DNA instead. Refocusing your efforts on mitochondria also works for especially degraded samples.

Keep samples on ice, and use as soon as possible.The rate of DNA extraction from Neanderthal bones barely exceeds 1%—2% of the genetic sequence, whereas the rate from wooly mammoths can be as much as 60%—80%. This happens because mammoths lived in colder climates and were frequently buried in ice, which inhibited microbial growth, notes Rubin. The same observation can be applied to other samples. (As for finding Jurassic DNA preserved in amber, though, don't count on it, says Rubin.)

Make your own buffers.Yale University's Octavian Henegariu sometimes does thousands of reactions per month, and he doesn't like surprises. He makes his own buffers in large quantities and freezes them in aliquots. Then, "if something happens I can easily troubleshoot it," he says. "If I buy from one manufacturer one week and from the other the next week, and stuff changes, I have no idea if it's me, if it's the DNA, if it's the RNA, or what it is." His own stock provides consistency and stability, he says, and also cuts costs dramatically. "Then I buy the cheapest Taq I can find."

Use a master mix.If you worry about the integrity of target samples as Noreen Tuross of Harvard University does, you want to minimize exposure at all stages of the PCR process. One way to do this would be to put a hot-start Taq into a master mix in the first place, just in case it's ultimately needed. "You're not doing multiple additions to the tube, and that helps with contamination [issues] a lot," she says.

Try adding BSA.Henegariu routinely adds a small amount of BSA to his PCRs to help stabilize the Taq. He speculates that "adding protein in there probably somehow attracts some oxidative radical species that are being produced during the reaction." Whatever the reason, he says, "it helps what I do."