Hello everyone , I'm working on microsatellite analysis of Saccharomyces cerevisiae. I started with ScAAT1 locus. Here is the raw data of the first isolate shown on the chromatograph (DNA Size Standard in red and PCR products in blue). My question is to know first if I have to consider all bands as alleles and then what sofware is appropiate for this kind of analysis.
With microsatellite regions that have been optimized you should only get one peak (homozygous) or two peaks (heterozygous). If you have multiple products you are looking at non-specific binding. You need to go back and optimize the PCR conditions so you get a single product.
Anna Schwabe, Thank you for your suggestions, I will repeat the experiment by increasing annealing tempearture during the PCR step and I will keep you informed of the result.
You can do a temperature gradient to maximize time. When I am optimizing I use a gradient between 47 and 65 degrees celcius. You can also mess with the magnesium (between 0.5-4.0 uL in a 12 uL reaction volume). Beyond that you can mess with DNA or primer volume also.
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