Me and my teacher try to amplify the microsatellite in seagrass (Enhalus acoroides) but sometimes the band not appear in the electrophoresis. Firstly we thought that maybe it caused by low quality and low purification of our dna sample. first we use CTAB for extraction and then we try to use Qiagen. The result was excellent in DNA purity shown by clear band and less frequency of smear. however, sometimes the microsatellite in some samples are failed to amplify and it doesn't appear in the electrophoresis. It just like ghost. could anyone tell me what kind of possibility for the failure which can be happen in this process?
Are the microsatellites that you are trying to amplify designed specifically for Enhalus acoroides? Maybe your samples are missing the proper binding region or you could have some sort of mutation in the binding section? You mention that the DNA purity was excellent; have you quantified this? It could be possible that you have too high a concentration of DNA in your reactions?
I don't know if this is what is happening in your case, but sometimes with microsattelites you get what are called null alleles - these are variants that do not amplify, probably because of mutations at the binding sites of the primers. If they're at low frequency they'll show up as an excess of homozygotes in your population, because only one allele is successfully amplifying in some samples. Your amplification failures could be instances of individuals who are homozygous for the null allele. Is this a well characterized system or are these loci that you are developing? Because if this is a locus that has been used in other studies then there should be some hint of null alleles in other data sets.
Unfortunately it could also simply be a problem with amplification, as Karl Lamothe mentioned - the most common error with PCR is too much template DNA, so trying dilutions of your template may help (1:10, 1:100, and so on - I've occasionally needed to go to 1:10,000). You could also try doing a positive control - try amplifying some other locus using your template just to check that the quality is as good as you think. If you have multiple microsatelilite loci and only one or two fail to amplify that's usually a sign that it's something about the locus itself. If nothing, or very little, amplifies, it's more likely to be a problem with your template quality.
The microsatellite primer was design specifically for Enhalus acoroides. Yes, the quality of DNA was quantified using the DNA ladder with known concentration. We have also try to dilute the sample which contain very high DNA but it's still cannot be amplified. Maybe the reason of the mutation at the binding site can be the one of the possible answer for that but I need to check it first for make sure.
We use the multiple microsatellite loci for this analysis but as I mentioned before that sometimes it just like a ghost.
by the way, thanks for the answer Karl and Laura :)
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