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Problems scoring microsatellite fragments

Problems scoring microsatellite fragments - PCR - Polymerase Chain Reaction Forum

Problems scoring microsatellite fragments - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 08-13-2010, 12:12 PM
Pipette Filler
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Unhappy Problems scoring microsatellite fragments



Hi, I am currently amplifying a number of microsatellite loci using fluorescently labelled primers and have a number of DNA samples that keep producing fragments that are difficult to score. These fragments appear to show two alleles with one peak of high fluorescence (usually around 7000 units) and another far smaller peak (usually around 1000 units). Given the height of the first peak, I am often not sure if the second peak is actually an allele or represents contamination. I have tried reducing overall peak height for these samples by using a smaller amount of primer or template. This has only produced equally sized peaks for a few of the problem samples. I was therefore wondering if anyone has any suggestions as to how this problem could be overcome.

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fluorescence intensity , fragments , microsatellite , primers , problems , scoring


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