MSP U primers very difficult to amplify... - (Aug/04/2011 )
We're having some problems with getting the U primers for our MSP to amplify. The M primers have nice bright bands and don't have a problem but the U bands hardly work and when they do, the bands are really weak. The primers are designed by MethPrimer and/or published sequences.
Can anyone give me any tips?
sorry to be a little naive,
but if your target is methylated then the U primers won't work anyway. Have you checked this?
Sorry,meant to say that we are trying to optimise the pcr using commercial unmethylated dna so the primers should work beautifully.
okay so you might need to lower your Tm in your PCR strategy to facilitate annealing of the U primer which tends to have a lower Tm if the same length as the M primer. (lower GC content).
This would mean that your U and M PCR cycling conditions would need to be different. To increase the Tm of the U primer I would suggest lengthening it.
these primer sets were published (and checked that they were on the right site) and we have tried all sort of optimising conditions, quite often I just get a very very faint U band