real time PCR primer for methylation - (Mar/14/2005 )
I am trying to design primers for H19 by using primer express, because I want to use probe in my real time pcr. But the software doesn't give me anything. If I use sybergreen, the primers I am using right now have dimer, so it won't work. Anybody can give me some suggestions?
I would like to ask one question, are you trying to design methylation specific PCR primers for a template that has been bisulfite treated? Or are you designing normal primers to the H19 gene, so you amplfiy off restriction digested genomic DNA?
I ask this because I don't think Primer express can design methylation specific primers. Check out http://www.protocol-online.org/forums/inde...?showtopic=4589 for specific primer designing tools.
I assume you would like to use the Taqman system, again I don't think Primer express can do this easily. However, you can "mock" bisulfite convert your sequence for primer express to pick appropriate primer and probe sets.
Thanks for your suggestion. I am doing methylation PCR for bisulfite treated DNA. I input the bisulfite treated sequece to primer express, and got nothing.