MSP is not reliable! - methodology discuss. (Jun/11/2008 )

By BSP,I picked a all methylated clone and a all unmthylated clone. Then I used M primers and U primers to amplify 2 clones at the same time. "m" and "u" band both appeared in M clone and U clone!
Contamination is impossible bacause I perform COBRA to verify the results. U products amplifying M clone with U primers was restriction digested using hhaI(site:gcgc),results was positive! Mismatch during PCR?It's strange.Any discussion is welcome.

I am not sure if I express myself properly,it's somewhat confused, and my English is poor.To verify the resluts,I perform MSP with another MSP primers designed by Methyl Primer Express.U clone amplified with M primers was negative,but the M clone with U primers still positive. Any suggestion is appreciated.

Try increasing the Tm to increase the specificity, you are getting non-specific amplification i think.

Nick

thanks for all the comments.The Tm was 55'C,I will try 60'C.But I don't think 55'C is low enough leading to non-specific amplification.I will return results.