Question about bisulfite PCR - (Jun/28/2006 )
In many papers I see people say that they used a specific amount of bisulfite treated DNA, typically 50-100 ng, for their first round pcr amplification. As I understand it however, it isn't possible to get reliable spec readings or even run the single stranded DNA on a gel to obtain concentration information. What would be the best way to estimate the amount of DNA you have after bisulfite treatment?
another hurdle in BiS treatment.
i usually go by volume and assume i have a certain amount. When i absolutely positively must know, i use QPCR and extrapolate the concentration. If you have a real time machine, or plate reader, this method is fairly accurate.
I also agree with sneth,
I hardly quantify my bisulfite treated DNA, if I say start with 2ug of gDNA for bisulfite and estimates that 90% of your DNA is degraded in the process, I would end up with 200ng or thereabouts (in 50ul). of which I use 4-10ng for a PCR (1ul for PCR).
it's usually volumes I work with also.