texasepigenetics

Hello all! Have loved reading this forum, always so helpful.

I have recently joined a lab and am picking up a former member's dna methylation project. The previous member successfully amplified DNA from tissue, bisulfite converted and PCR amplified regions of interest. These regions were then sequenced. My job is to go back and confirm her results by hand before we can publish. Easy right? Wrong!

I cannot get her primers to work! I am isolating DNA from the same tissue. I am using the same kit to bisulfite convert (Zymo methyl gold). Same primers, same taq polymerase, same PCR cocktail. I've been running her primers on DNA from tissue (of "unknown" methylation status) and on DNA from the tissue that I have treated with M.SssI to fully methylate. I am sometimes able to get the correct size band, but only in the fully methylated DNA, never from the DNA from that tissue that has not been treated with M.SssI.

Now then, my helpful colleagues... where should I assume my hang up is? I know the primers "work," since the previous person was able to get a product from bisulfite DNA of unknown methylation status. I am thinking that perhaps my bisulfite conversion is not working efficiently, is that most likely? (Since the fully methylated DNA would not require as much "conversion power" from the reaction to convert the DNA?) Or is it possible the the M.SssI treatment and/or NEB buffer that I'm using is doing something to that DNA to increase the efficiency of the conversion reaction? Any and all ideas will be welcomed!