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