global DNA methylation quiagen kit - (Oct/26/2009 )

we have had quite a bit of variation in measurements and in between replicate wells for measurements of global DNA methylation.

This can be reduced with accurate pippetting and pippetting times (or using a robot to dispense the reagents). Because this can lead to some of the variations you maybe seeing.

What don't really like about this protocol is you are relying on faith that the methylation standard given in the kit is truly 100% methylated. Also the measurements you make on your test samples will always be relative to the standard you use from the kit, it is difficult to get a precise measure of what is actually going on in your sample, it's relative to the standard.

So we have not focussed any further efforts in working this protocol up fully, there are other methods that are less variable than this kit.

Nick

methylnick on May 18 2010, 05:30 PM said:

Thank you for your opinion, could you tell me which method are you using that is better?

Hello everybody, PLZ help me,

I was working on CpG methylation stuff, and from your guides, especially MethylNick, I finally could get very nice bands, my advisor and me became sooo happy and and I introduced your site to him too, we both thought that the most part of the work has been done, but... sad.gif .but I can not get any sequencing result from PCR product,,, I like to tell a little more about my procedure;

I extracted PCR products by gel extraction kit (qiagen) and directly I took 9μl of that and 3.2μl of forward primer in one 0.5 ml tube and send to sequence, but just untreated samples(without any bisulfite treatment) had results. I thought maybe there is one agent in my samples whihc does not let sequencing does well job, so I washed my samples(before DNA is treated with bisulfite with zymo kit, then amplified by PCR, and PCR product was extracted by kit) then I put 9μl of that in to a 0.5 ml tube and I add 3.2μl of reverse primer instead of forward primer and sent for sequencing again, the same result, I mean no any sequencing result for bisulfite treated samples.

So, PLZ let me know what you think? it is important that my PCR products are very short, <200bp so if you think I have to amplify my PCR products before sending to sequence?

I'm disrupted too much, my great friends, PLZ give me your ideas,
Thanks

Hi everyone,
I used the sigma kit twice and it was really poor with no reproducibility. I am going to try the epigentek supersense kit!