what is the best dna methylation/modification kits? - newbie kit comparison (Jun/12/2007 )
Okay, I'm having some problems with the Qiagen kit. Ive got frozen paraffin embedded tissues and modified the extracted dna. i ran two pcr amplifications at about 300bp and then 200bp. dna recovery is low after the bisulfite modification. My samples are showing nothing! Any ideas?
What about PCR from the unmodified paraffin extracted DNA? Does that work?
And - forgive me for asking, but still - you did design the primers to reflect the modification (unmeth C -> T)?
I guess low recovery is normal. I only have experience with modified DNA, but what I gather I'm using rather bizarre PCR conditions, very high primer concentrations and Q Solution. You might have to optimize the PCR.
Qiagen kit worked in the end, but it didn't go as smooth as I wanted it to. A colleague of mine recommended Sigma Aldrich's new kit Imprint modification kit, so I'm going to try that for my next experiment. My colleague also ordered an EpiQuik whole cell bisulfite modification kit by Epigentek. Will keep you guys posted.
Also thanks for your help cyburn, i had it figured out, u were right about optimizing PCR, i overlooked some things
Also thanks for your help cyburn, i had it figured out, u were right about optimizing PCR, i overlooked some things
hey genoman,
have you figured that out? i'm trying to decide which kit to get since i've tried both qiagen and zymo and, as my modified primers, i'm not happy AT ALL. recovery is awful and i can't try more than 3-4 PCR reactions with each elution. i'm wasting kits and my precious DNA. i'm frustrated with the fact that i don't know how much DNA i should use for PCR and if that was the reason it didn't work. i've seen posts on the HGS kit (methyleasy) but it's expensive (shipping from australia [to US]). i haven't heard of this sigma kit and would appreciate any thought of yours.
i have a general question for all members and it might sound stupid since i'm new to that. i've done and a lotta you guys, BS-treated gDNA as template, but only the unmodified primers work. so i went forward and TOPO-cloned it. of course sequence came not converted but BS-treated this plasmid and sent for sequencing. now i'm happy to up to 80% of convertion. than comes my questions: DOES IT MAKE DIFFERENCE IF I CLONE BEFORE CONVERTING (SAVING MY FRUSTATION AND MY BOSS' MONEY)? DOES THE METHYLATION PATHERN CHANGES?
thank you,
doug
Hi Doug,
if you clone your fragment, the methylation information is likely to change. Your plasmid will be multiplied and some if not all of the information is likely to get lost. I wouldn't try that. But i can't really understand your experience, as we get top results with Qiagen and other kits. As Nick always says: NEVER TRUST YOUR PRIMERS (okay, that was not cited by word, please forgive me Nick 
but I think I got the point, no?)
K.
Hi Doug,
I would agree that primers and primer design is very important. We get best results with Methyleasy. Cost was originally an issue for us too, but now we use the newer version as it is much cheaper ad has a column purification step.
but I think I got the point, no?)Krumel,
indeed this is what I have been trying to tell people delving into methylation madness!!
As for Methyleasy, as methylman1 pointed out there is now a version 2.0 MEthyleasy called Methyleasy Xceed which is much cheaper, much easier and less hands on time, you can do bisulfite treatment in half a day now...the way technology is progressing astounds me!
Cheers
nick
Wow. I just tried Sigma's imprint dna modification kit, and must I say I am VERY impressed. I'm almost giddy with how easy it is compared to all the fuss I've put up with before. I highly recommend this kit to ANYONE who wants to save time with their methylation analysis. The datasheet instructions were multiple times easier for me than working with qiagen's cumbersome steps. I'm not always the keenest person when it comes to bisulfite modification but amazingly enough I got it working on my first run through. My yield was way greater than qiagens, using qpcr
Plus i only had 0.5 ng of dna to work with -- amazing... Everyone use this kit if you want to make life easier on yourself!
Hi hi,
I am using the EZ DNA methylation kit and it works fine. You loose about 20% of the gDNA, but it is fairly robust.
I am however not very content with their "Gold" edition or the alternative 'cycling protocol'. Especially the cycling protocol looks nice and similar for a whole amplicon, but when you look quantitively (Mass Spec method) you find that individual CpG sites are sometimes not completely converted....Just follow the protocol with overnight BS conversion...It works like a charm
Greetings from a gloomy holland
ET2B