minimum amount of dna for DNA-conversion - (Oct/09/2006 )
i´m using the zymo ez kit and so fare i got excellent results for my pcr from cell culture samples. now i´m trying to an analyze my patient samples - the problem is that i only have a limited amount of dna ! what is the minimum amount of dna for the DNA-conversion ? is 10 ng enough ? or 100 ng ? .... the manuel says 500 pg - but i don´t believe this !
any help would be great - thanks !
one thing to look out for scrapie is that although the Zymo kit can convert 500pg of gDNA it would only be enough for one or two PCR reactions as from memory you resuspend in minimal buffer and use most of it for the PCR after conversion. This is because bisulfite also degrades your DNA and up to 90% of the starting amount is lost in the degradation/conversion process. Check with the instructions from the Zymo kit to see how many PCRs can be performed with such an amount and go from there.
I have said this many times, MethylEasy seems to do a better job in that no degradation occurs during the conversion process and this seems to be far more superior than Zymo's or chemicon's kit.
I typically use 500ng as a "safe" amount, I have been using samples where there were minimal amounts between 20-200ng, with the nature of the sample it's difficult to measure the concentration, but I am able to obtain products from it.