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[spjgjsm]

Hi

I am currently working up some MSP assays. I designed the primers using methylprimer. Due to a shortage of bisulfite-treated DNA I haven't been able to fully optimise the PCRs, but at first sight everything seems OK with sibgle bands using an annealing temp of 55C [is this a good temp to start at??]. My other PCR condidiotns I used are hot-start and 34 cycles.

Problem is, I also get weak bands when running non-treated DNA samples - surely I should get none in these. Furthermore, when using universally methylated DNA controls, i get bands in the un-methylated PCR as well as the methylated PCR.  And the results are not 100% reliable - i.e. when i run the markers on the same set of samples on another day i get different band patterns. I think this is likely to be a problem of primer annealing and specificity - what can i do to tweak my reaction so that the primers only amplify where they should....

Cheers

Jon

[pcrman]

Hi Jon,

Although non-specific amplification is a problem with MSP, it is uncommon to see band even with untreated DNA, because the primers are designed based on the modified sequence which is almost totally different from the original sequence. Primers are usually picked at places where they contain stretch of non-cpg "C" to discriminate modified DNA against unmodified or uncomplete modified DNA. You should check if contamination is the problem.

55C is good temp to start at, try raise it a bit to see if that can get rid of the non-specific amplification.