Sigma Imprint DNA methylation quantification assay kit - (Sep/20/2013 )
I am *trying* to use the above kit to quantify the amount of 5mC in the genome of 32 samples, of 2 different genotypes and 2 different timepoints of viral infection.
Although I have found a very nice consistent pattern of differential global methylation between both the genotypes and timepoints, I am having difficulties with high levels of background in my blanks, so can't really trust my data.
My blanks are reading up to 0.7, when they need to be <0.1! I have tried doing a serial dilution of the DNA Binding Solution (the reagent I'm using as a blank) and I got down a blank reading of 0.5 for the lowest dilution of DBS.
I was wondering whether anybody had any experience using this kit? I know there's one other thread that I've managed to find -
http://www.protocol-online.org/forums/topic/15184-global-methylation-assay-kit/ - from 3 years ago, so I'm hoping there are other people out there who have troubleshooted this assay.
Basically, I think I've narrowed the problem down to it being either inadequate wash steps or too long colour development time (although I have followed the Sigma protocol directly on both accounts). I was hoping that somebody would be able to advise me on how long they left the developing solution on, as I've been leaving it for up to 10mins (too long?) and how many wash steps they may have added, if any.
Thanks very much
Never used this kit. I believe you have included controls (methylated or demethylated DNA), how do the controls look like? Since the kit bases on antibody reaction, inadequate wash could invariably contribute to high background problems.
Hi, thanks so much for replying :) they give you a methylated DNA control, which you can either use to generate a dilution series (i.e. standard curve) or just use once as a single-point quantity. I did both, but both times I did the dilutions series my results are WAY out. These are my data for my most recent optimisation experiment:
WELL ID raw data A01 200ng control - diln series 1 1.328 B01 100ng control - diln series 2 1.472 C01 50ng control - diln series 3 1.496 D01 25ng control - diln series 4 1.296 E01 100% DNA binding soln - blank1 0.633 F01 50% DNA binding soln - blank2 0.597 G01 25% DNA binding soln - blank3 0.57 H01 12.5% DNA binding soln - blank4 0.601
A01-D01 being my control (did a dilution series from 200ng down to 25ng); E01-H01 were supposed to be blanks...! And I like to think I know my way round a lab by now and don't cross-contaminate (for the wash steps I aspirated separately from each well, rather than just turning the plate upside down) or confuse my samples.
I followed the protocol exactly - the recommended # wash steps etc, and <10mins development time....
Think I will try a test this week where I do extra washes for every sample, and leave well 1 for 2mins development, well 2 for 3 mins development etc etc. Hopefully I will get to the bottom of this!! As I said in my last post, I'm finding a consistent pattern in my samples, but I can't really go any further with a blank and standard curve that look like that!!
I've used this kit before a few years ago. I had similar problems with high background signals. To get a lower background you need to increase number of washes. As far as I can recall, my development time was more around 2 minutes, not 10.
Anyway, my lab has since switched over to the methylflash global dna methylation kit a while back and we don't have any problems anymore with the background. http://www.epigentek.com/catalog/methylflash-methylated-dna-quantification-kit-colorimetric-p-2094.html
It's a very similar assay. Good luck