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Home page > MLVA Web service > Frequently asked questions > I can get the PCR assay to work, but am having problems analyzing the bands to get accurate sizes from my gels. I wonder how to actually calculate an exact band size and the repeat number. I am using the size markers to make a standard curve based on distance migrated on the gel. This old fashioned method is working, but once I get a band size, I cannot figure out how to get a repeat copy number.

I can get the PCR assay to work, but am having problems analyzing the bands to get accurate sizes from my gels. I wonder how to actually calculate an exact band size and the repeat number. I am using the size markers to make a standard curve based on distance migrated on the gel. This old fashioned method is working, but once I get a band size, I cannot figure out how to get a repeat copy number.

When setting up the assay, a first step is to identify a reference strain. Any strain in current use in the laboratory is adequate. Vaccine strains are a good choice since typing data for them is given in the Le Flèche 2006 report (see supplementary data file). Then eventually it will be useful to sequence the alleles from this reference strain, to make sure no discrepancy will have been introduced. As described by Garcia-Yoldi et al., 2007, some loci may have mutated upon culturing in different laboratories, especially among panel 2B. Once this has been done, conversion from allele size to repeat copy number does not require precise sizing. The only requirement is to be able to confidently distinguish an allele from the next larger and next smaller. The allele conversion table provided in the utilities is essential for this purpose. There is no need to make standard curves etc.