I am working on varietal identification of black pepper varieties using SSR markers. After running PAGE I am getting bands other than the expected amplicon size of 200 bp. I am getting bands of size 600 bp, very prominent bands. Can we count them as alleles. Black pepper is a diploid plant
I guess you are getting non-specific bands. Please re-consider your PCR conditions including DNA template concentration, primer concentration and annealing temperature.
Hey! I think you cannot count the 600 bp as another allele because it might be an unspecific band as mentioned before. Allelic variation of SSRs is given by a increase or decrease in the amount of the repeated sequences... so the differences in size are usually not that big. You can also check in literature the range of variation in size of the alleles to see what differences might you expect. Therefore, you may score the bands close to 200 bp.
Hi,
I would suggest you to increase the specificity of your PCR. Try with a gradient PCR and then observe the results. if the 600bp persist you might think to send it for sequencing.
Best regards
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