Contamination/no amplification in MSAP-PCR - (Nov/11/2005 )

Hi!

I am working on DNA methylation using MSAP-PCR but have the same recurring problem and I find it quite confusing. I always get two different patterns of PCR products:

1- Whether I get more amplification in my negative control than in my test
2- Whether I don't get any amplification anywhere

Someone in the lab who is not working with MSAP-PCR but who is also doing low stringency PCR systematically gets the same results at the same time, on same days.
We both are working with different goodies, so the reagents as well as manipulation problems don't seem to be in fault.

Has anyone ever experienced this kind of situation?
Thanking you in advance for your kind collaboration.

that's strange

ar eyou both using the same pcr machine? there maybe something in the air that's contaminating your reactions if you are getting the same products.

I am not convinced it is not a reagent/manipulation issue. Change everything, water, taq, buffer, tips....everything!! Bleach and ethanol your benches. contamination is something that is very difficult to get rid of.

Actually, we are both using two different machines each! And we already changed all the reagents, cleaned the pipettor barrels, used freshly autoclaved PCR tubes...

In the past 2 days, none of us got amplification in the negative control, but I still don't get amplification in my test. Since MSAP is a low stringency reaction, I am hoping that no amplification in negative control really means there is no contaminant DNA to amplify and that the no amplification in the test means my template DNA is just being picky. So I am going on trying to get bands in my test (since it is CG rich, I will use longer pre-denaturation time, might try DMSO, different dilutions of template DNA and primer...).

Thanks for your help! Hoping to provide some answers soon about that awkward situation...

if that's the case, you may need to optimise your pcr conditions. Mg2+ titration would be a start as well as a gradient Tm.

N