4 replies to this topic

[PostDocTrauma]

Hi all,

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?

Edited by PostDocTrauma, 04 August 2011 - 02:08 PM.

[methylnick]

sorry to be a little naive,

but if your target is methylated then the U primers won't work anyway. Have you checked this?

Edited by methylnick, 07 August 2011 - 06:38 PM.
typo, should be "methylated"

[PostDocTrauma]

Sorry,meant to say that we are trying to optimise the pcr using commercial unmethylated dna so the primers should work beautifully.

[methylnick]

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.

[PostDocTrauma]

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