Methyl Primer Express from ABI - (Nov/04/2006 )
Just like to ask if anyone have already used the new MethylPrimer Express from ABI. Any reviews about the primers the program designed? Is it good?
By the way, I've always had this question in mind. Are there any quick way of determining the best temperature a pair of MSP or BGS primers will work without going through the tedious way of optimizing them by PCR and subsequently running a gel??? Any program to calculate the best temperature to use or some general rule of the thumb from the Tm something like that? Any advice??
MethylPrimer Express: I have used it for different genes, and it worked in more than half of the cases. That's definitely better than for example MethPrimer. It helps a lot to manually check the primers that MethylPrimer Express suggests, but then they should work. As I always use nested PCR, I use the modified sequence of the product (you'll find it on the output page) and design second round primers with this sequence in Primer 3. That works fairly well for me.
Annealing temperature - the thumb rule, 3 to 5 degrees below Tm seems to work also for methylated DNA, but you should be aware that you have to use the exact conditions for both primer sets in MSP, otherwise you cannot compare the CT values! I would always prefer to invest the PCR and gel runs to optimize my products...
Much luck for your experiments!
Look at the pinned thread on top about MPE program