MSP: M and U primers products - (Aug/09/2007 )
I am new to the methylation PCR. For my gene which has a high CpG content in the exon 2, I have picked two primer set using Methprimer. For my understanding when M amplifies the region that is methylated and when U amplfies it is the opposite. I have put the jel picture, which confused me. I have bands for both, but M primer amplicons are stronger. By the way the I used the EZ methylation kit. I realized that ı dont seem to have a proper control. Which is the best strategy for the next move?
thank you for your time and help,
this is not uncommon. Try to use fully methylated and unmethylated DNA as control. Try using unmodified DNA to control if your primers are specific for converted DNA.
Your samples might contain a mixture of heterogeneous cells. If you need to quantiy the methylation, you can use real-time MSP or more appropriate methods like BSP or Pyrosequencing.