Was wondering if anyone can help me with my MSP.
I used methprimer to design primers for a bunch of genes. I used the Tm from metprimer which ranged from 55-59 degrees. To use the same PCR I used an annealing temp of 50 for 40 cycles.
I am getting single bands at the right size but my U primers and M are picking are not discriminating between unmethylated and methylated DNA.
I used Qiagen control M and U DNA and some cell lines. My unmodified DNA is not being amplified and primers to unconverted DNA are picking up bisulfite converted DNA. I am using EZ DNA methylation kit and treating 500 ng.
What could be going on? Could annealing temp have anything to do with this?
Edited by Boba, 20 April 2010 - 07:40 PM.