Hi Michael,

Thank you for your answer! There is no information provided regarding that preference of the antibody for ssDNA but the instructions advise to begin with purified genomic DNA with no comment about denaturing. So I think I'm OK there....

Regarding the positive control DNA - it was this DNA that I used to generate the standard curve so yes I have the same problem with it in terms of technical replicate variation.

I am further investigating whether our samples are suitable. It may indeed be that the levels of methylation are too low for this particular kit to detect. Given that I have tried everything else - this could be it. Whatever I have tried it seems that I cant get the samples above the background level.

Thanks again for your useful comments - a couple of things in there I hadn't thought about!

Cheers,

Nicki

Hi Michael,

Could you please supply the protocol for dot-blot technique. I want to compare the DNA methylation between two samples. I dot the denatured DNA on positively charged nylon but I am not sure about the following procedures.

your sincerely!

Yu Yin

Hi Nicola,

I've employed Zymoresearch 5-mC DNA ELISA Kit sucessfully in samples with low total DNA methylation. Depending on your desing make sure to include enough replicates.

Hi Yin,

I've used the protocol in the attached publication for dot-blot.

Article Dynamic regulation of 5-hydroxymethylcytosine in mouse ES ce...

Hi Nicola

Recently, I have been used this kit to explore the global methylation in my cancer cell line. Anyway, I think it works for my sample. Could you provide more information that what your negative control is? Since it's difficult to choose the negative control that without methylated DNA completely, I'm not sure how you prepare you DNA sample and you need to make sure that DNA preparation method didn't affect to global methylation of your sample. 

I have since found out that this kit actually cant detect the level of methylation in my samples!! I have switched to the more sensitive EpiGentek MethylFlash Kit and am having far more success. Thanks for all your answers! 

Can I ask what level of variation between technical replicates people are seeing with their samples?

Many thanks,

Nicki

Hi Nicola

Am currently using the Sigma Aldrich kit, however my problem is more of opposite to your problem. I'm having too high %methylation for my samples most of which is around 50%. May I ask your source of DNA and method of DNA extraction? maybe it has an effect. My source of DNA is human blood and the method of extraction was the salt extraction.

Again, do you know any publication(s) which has used the Sigma Aldrich kit in their method?

Thanks,

Tinashe

Hi Tinashe,

The DNA I used was from insect tissue extracted using the QIAgen DNeasy Blood and Tissue Kit with very minor changes to manufacturers instructions. Are the 260/280 ratios for your DNA around 1.8 or higher?

I will have a look tomorrow when I get to the office for any publications I found while using this technique that might be of use to you.

Nicki

Hi every body

I'm using the "Imprint DNA Methylation Quantitation Kit" and I have the a problems as Nicola Cook faced which is getting a background readings (negative control readings) that are actually higher than that of my DNA-containing samples, even I can not detect any color development in any of my sample replicates

Unfortunately,  I can't not shift to a new kit now.

Does anybody has a new suggestion?

Hello,

Here you can find a comparison of four different ELISA-based DNA methylation kits:

https://www.karger.com/Article/Abstract/486201

For low 5mc amounts (

Hi,

I have some promlembs about Zymo 5 mC DNA ELISA Kit. (D5325)

It does not give any results.

I used on this kit MSG (monosodyum glutamate) injected rats as material.

Would you share if there is an information about this Zymo Kit's?

Many thanks.

Dear Rafig Gurbanov ,

I am planning on using this new kit from Epigentek that you recommend (MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit). Have you already used it? How's been your overall experience? Is the variation between replicates solved?

Many thanks in advance.

Hi David,

I did not have much success with this kit for our experimental purposes. The MethylFlash kit was indeed the best one I tried. However, our organism only has c1% methylation across the whole genome and the variation in technical replicates was generating too much noise for the VERY small changes in methylation we were looking for. I had to move away from ELISA-based methods altogether for our purposes and use BS-seq instead.

Hope that helps!

Nicki

Dear Nicola Cook ,

Thank you very much for your valuable reply. Can I ask waht is that organism you are working with? I am working with a human cell line so I am not really sure how the kit performs with such samples.

Best regards,

David