I am *trying* to use the above kit to quantify the amount of 5mC in the genome of 32 samples, of 2 different genotypes and 2 different timepoints of viral infection.
Although I have found a very nice consistent pattern of differential global methylation between both the genotypes and timepoints, I am having difficulties with high levels of background in my blanks, so can't really trust my data.
My blanks are reading up to 0.7, when they need to be <0.1! I have tried doing a serial dilution of the DNA Binding Solution (the reagent I'm using as a blank) and I got down a blank reading of 0.5 for the lowest dilution of DBS.
I was wondering whether anybody had any experience using this kit? I know there's one other thread that I've managed to find -
http://www.protocol-...tion-assay-kit/ - from 3 years ago, so I'm hoping there are other people out there who have troubleshooted this assay.
Basically, I think I've narrowed the problem down to it being either inadequate wash steps or too long colour development time (although I have followed the Sigma protocol directly on both accounts). I was hoping that somebody would be able to advise me on how long they left the developing solution on, as I've been leaving it for up to 10mins (too long?) and how many wash steps they may have added, if any.
Thanks very much