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

Hi,
I want to detect global methylation using quiagen kit but have problems. After trying in the way the protocol says, I did not get signal in the samples (less than 0,2 O.D) and very low for the methylated DNA control that it is supplied in the kit. I would appreciate very much if somebody has used it and could tell me what can be wrong. In my experiement I used 50ng of the methylated DNA control and the same for my samples. I could try more. Is it also worth trying to use more concentrated antibodies (I diluted them 1:1000, as it is advised in the protocol). For preparing the samples I just diluted DNA in the binding buffer. Is it worth trying to dry DNA before?

Thanks a lot

I am sure there is a trouble shooting section in the manual you could try out.

I haven't tried the qiagen kit, I have tried the epigentek protocol and get the reverse (Saturation well before the reccommended time).

If it's ELISA based, maybe you can let the development of the colour go a little longer, (for epigentek it's 2 minutes) before putting in the stop solution?

methylnick on Oct 26 2009, 01:31 PM said:

Thanks for your answer!
I made a mistake with the kit comercial, it was from sigma.
I´ll check protocol for epigentek also. Did you manage to reduce saturation reducing development tim?

bcsein on Oct 27 2009, 06:06 AM said:

sure did, 30 seconds was enough to ensure linearity. another way was to reduce the antibody concentration. which would be opposite to what you are after.

Nick

methylnick on Oct 29 2009, 02:46 AM said:

Thanks a lot

Hi guys, I'm new here and just registered when I saw this post. I hope maybe someone can give me some pointers:

I'm also using Sigma's Imprint kit for global dna methylation measurement, but am having some trouble getting good results because my OD for blank is constantly high. In a gist:

The first time during color development, the color started getting black almost instantly within 30 seconds. I was still told to shorten the color development time, so I shortened it to just 15 seconds before it started getting black precipitates. The reaction is way too strong and quick! I was also given suggestions to increase washing time, which I've did too (washing vigorously!). The OD for my positive control is very good though. I am lost.

Also, are there any other commercial kits out there that might be good to try (I know Methylnick mentioned an epigentek protocol but I'm not familiar with the company, but I see they offer some pretty interesting options)?? I'm almost running out of reagents here and I dont want to spend more of our budget for something that might not work. Help!

Thanks ahead of time

I believe that the epigentek kit is now the Sigma kit. I maybe wrong though.

racingstud on Jan 21 2010, 10:15 AM said:

Yeah I was told this when I called Epigentek in early January. Kudos to them for giving me a trial kit for their fluorescent version (Supersense kit), which worked very well. Thank goodness because I was running out of my precious samples. I definitely plan on buying their full kits in the future.

epigenius on Jan 22 2010, 09:55 AM said:

Hello, I am using the SIGMA methylation kit for whole genome. I have many problems. Results for the hypermthylated control that is in the kit , are changing a lot from assay to assay. I read te Epigentek protocol and the solutions and assay were so similar that I thought it was exactly the same, but it wasn´t because I asked the technique service.
I also have the problem that there is a high OD in the blank (only binding solution), and it is also changing from experiment to experiment. I would like to know if anybody has got good results with this kit, or with the EpigenteK kit, so may be it is worth changing. The technical service adviced me to dry the wells very good after washings , (this is not coming in the technical bulletin of the product, but it is very IMPORTANT). Even doing this the results are very bad.
I would like to know your opinion.

bcsein on May 13 2010, 10:59 AM said:

do u have a fluorescence microplate reader? im getting good results with Epigentek's Supersense kit