I have a problem with the M13 tailed primer method - M13 tailed primers-SSR (plants) (Oct/24/2007 )

Dear All,

I hope this message finds you well. I have been working with the M13 tailed primer method using the IRDye (LICOR) for microsatellite assessment of diversity in an important crop. The SSR markers that I chosed have been previously tested with standard PAGE (silver staining). The resulting PCR products with flourescence had the expected inheritance patterns, but the product size is higher than expected. The tail that is in the forward primer has 19 bp, but the differences between the Silver PAGE and Flourescence using the same controls for both, shows inconsistencies in the molecular weight (I mean, molecular size range since 4 bp to 40). I need to make the right calibration of my band sizes with respect to the silver ones, because I want to find the genetic relationships of my cultivars compare to those that are in big collection, that was previously screened for thousands of plants with the same SSR markers using standard PAGE.

Thanks for your help and interest,

anacorrea