Can't get MSP PCR products at all after treatment! - Help with MSP using Chemicon Fast DNA Mod Kit (Feb/20/2006 )
I need some help with MSP using this Chemicon Kit.
I have been following the protocol they provide and I cannot get any PCR products at all. I am using cell lines that I have extracted using Laried Lysis. I then tested this DNA using other primers ( before bisulfite treatment) and it looked good. I then treated the DNA per manufactures protocol and end up without any bands.
I know that my primers work, because I tested my IVD methylated control before I started other samples. It worked then, but I cannot get it to work again. It seems that my DNA is being degraded during treatment. I only let it sit for the min specified time of 16 hrs. I don't know what else do try. All my solutions are perpared fresh and I use sterile nuclease free water of course. Help!!
does the kit come with a working control....ie: gDNA and a primer set that you convert and PCR along with your samples? if so, did this work?
Also, you will get degradation within the conversion with the exception of MethylEasy....that I am aware of.
Failure in PCR could mean your primers are not efficiently amplifying converted template.
You could try lowering the annealing temp of your PCR step and see what happens. Also you may need to perform a generic first round of PCR with non-specific primer sets and then perform your MSP in a second round.
Can you post your sequence and primers so we can all have a look at them?