Invitrogen Methyl Miner Tips - (Mar/10/2013 )
I'm wondering if anyone has experience using the Methyl Miner kit, and if so could you offer any tips not mentioned in the protocol??
For instance, has anyone found loading a certain amount of DNA provides the best results? I was planning on loading ~500ng I'm also curious how much I can expect to elute from the 500ng I start with?
All tips and suggestions would be much appreciated, as I don't have much time to spend for optimizing the protocol...
Someone has to have used this kit....Anyone?
Hey, Hockey! I realize that by now this assay is old news for you, but I've just found it!
I'm using the kit now in order to create libraries for sequencing from the enriched fractions.
Based on Bioanalyzer results, I've been able to capture and subsequently amplify fractions from as little as 100 ng DNA of starting material for the Methyl Miner.
What has your experience been? How low have you been able to go for the Methyl Miner?
A couple of concerns I still have are that I'm weary of any attempt to reliably quantify the DNA in the captured fraction. Nanodrop will overestimate your concentrations, and the Qubit (even with the High Sensitivity kit) gives results which aren't repeatable enough for my satisfaction. Ultimately, I think the Qubit is better in that it should be able to tell you "yes" or "no" about if you have captured DNA, but that the number it yields has a huge error (> +/- 10%).
Since it's been months since you posted this, I guess you probably don't need help anymore.
However, I've been able to use the Methyl Miner protocol with as little as 100 ng of input DNA.
I'm hesitant to trust the values you get from Nanodrop if they're below about 20 ng/uL. In my hands, the Nanodrop grossly overestimates DNA concentrations below that threshold, and it is especially prone to do so if you've dissolved your DNA in anything other than 100% pure water.