Several people expressed an interest in hearing the responses I got to my microsatellite question. My original question asked for help with the following two problems with microsatellites I had developed for frogs: (1) the microsats don't amplify consistently (i.e. one sample may give nothing for one PCR but work beautifully the next time under the same conditions), and (2) my results are largely not reproducible among PCRs (i.e. I get different peak sizes in successive PCRs of the same individual a large percentage of the time). These problems happen for all 8 of the microsats I developed. Here are the answers I got: - You sometimes have drop out of alleles (especially the longer ones) and also some loci that differed by one allele between runs due to A-overhangs. You can prevent by adding a 30 min step at around 60 degrees to the PCR, but I'm not sure about the allele drop out. - Amphibian microstatellies are just difficult. Some have had luck chelexing their already extracted DNAs. - Check your primer sequences again and try to redesign primers. As well try to standardize your PCR with lower annealing temperatures. - inconsistant amplification is mostly a handling and/or contamination problem. Are you sure your conditions are always REALLY the same? Do you vortex/centrifuge all eppis after thawing? Could it be that you use different batches of water/buffer/plasticware? Try to use fresh batches, re-dilute your primers from stock. - different peak sizes: may occur due to in vitro slippage events (in fact, during your PCR the same effect happens that leads to polymorphism in nature: loops form during strand synthesis). This can be circumvented by optimization of PCR chemistry (try different Mg and dNTPs concentrations), choice of enzyme (try at least 2-3 different brands to see which one works best with your template), not too many cycles (maximum 32, less are better; not more than needed for a clear signal on the capillary), and a long final extension at 68° (15 min). - With degraded DNA the low template number causes some alleles to be amplified over others, almost randomly. - I know of others who have had problems similar to yours on the ABI 3130 capillary machine. Many of the problems went away once formamide was used in the prep of the samples for loading into the sequencer. - Have you tried running the diagnostic tests on your PCR machine? If it's not heating to the same temperature consistently that could explain your problem. Likewise, if you use different PCR machines you could see a similar effect. Corinne L. Richards email: clrichar@umich.edu Ph.D. Student University of Michigan Museum of Zoology Division of Reptiles and Amphibians & Department of Ecology and Evolutionary Biology clrichar@umich.edu