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global DNA methylation quiagen kit


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14 replies to this topic

#1 bcsein

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Posted 26 October 2009 - 10:43 AM

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

#2 methylnick

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Posted 26 October 2009 - 12:31 PM

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?

#3 bcsein

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Posted 27 October 2009 - 05:06 AM

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?


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

#4 methylnick

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Posted 29 October 2009 - 01:46 AM

. Did you manage to reduce saturation reducing development tim?


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

#5 bcsein

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Posted 30 October 2009 - 11:10 AM

. Did you manage to reduce saturation reducing development tim?


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


Thanks a lot

#6 epigenius

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Posted 16 December 2009 - 08:48 AM

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

#7 racingstud

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Posted 21 January 2010 - 07:15 AM

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

#8 epigenius

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Posted 22 January 2010 - 08:55 AM

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


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.

#9 bcsein

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Posted 13 May 2010 - 06:59 AM

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


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.


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 wasnŽt 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.

#10 epigenius

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Posted 17 May 2010 - 08:41 AM

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


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.


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 wasnŽt 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.


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

#11 methylnick

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Posted 18 May 2010 - 05:30 PM

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

#12 bcsein

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Posted 26 May 2010 - 07:32 AM

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



#13 bcsein

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Posted 26 May 2010 - 07:34 AM

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

#14 epimaster

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Posted 27 May 2010 - 09:58 AM

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

#15 epicrazy

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Posted 09 July 2010 - 12:54 AM

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!




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