DNA Global Methylation with Epigentek kit-having trouble - (May/11/2014 )

My lab just ordered the Epigentek kit for global DNA methylation. We are using the standard curve reagents provided with the kit and have gotten a linear regression for our standards only twice out of many tries. We have figured out how to make our microplate reader shake the plate and have waited the required time(s) listed in the protocol before reading the plate, putting in different solutions...etc. I find that there is a big difference in the variability between duplicates and am not sure if a blank is needed or not? The protocol doesn't specify that one is needed but we have included one anyway. Does anyone have any tips on how to get this to work that newbees may not know?

Thanks,

enidfern

For your standard, are you taking only the most linear parts for calculation of the slope? You do not need to take all the concentration points as it eventually plataeaus out. As you're dealing with methylation and such small sample amounts, the reaction times of this kit is much faster and more sensitive than your traditional ELISA. That means you need to be very careful with pipetting, so use a multichannel pipette to add solutions into your wells simultaneously, or else you'll get exponentially heavier variation as the experiment progresses and especially during color development. Also, make sure you take note of when to change tips and don't let them touch the bottom of wells to avoid contamination (when appropriate). I think there's a demo video of the process on youtube or on their webpage. Also, I don't even bother using a microplate shaker. I just hold the frame against the flat surface, and shake it manually with the skillful execution of my hand and wrist :)