I am trying to genotype some short tandem repeats in a population, using fluorescently labeled M13 universal primer. I succeeded with 2 of the STRs but for the last 2, the M13 strategy does not seem to work. I've changed the Mg++ concentration, the primers ratio and concentration, the annealing temperature (standard PCR, gradient or touchdown). I've added DMSO, BSA... On the agarose gel I can see the band but running the genescan I don't get any signal, so I suppose that the M13 primer is no being incorporated... What do you think?
Strange after so many trials along with variation in reaction parameter you did not get any result. Look M13 primer are highly efficient and you might be having problem with template DNA concentration. So make one trials with altered template DNA in gradient PCR and I am sure you will have a positive result.
I'll check the gradient PCR, I think I haven't tried it yet. Regarding the DNA concentration, I've tried a wide range, so the problem is the incorporation of the last primer, the M13. Thank you Mahendra!
Where you able to incroporate the M13 primer to those last two STR?
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