methylation analysis - methylation analysis using epitect bisulfite kit from qiagen (Jan/15/2008 )
hi every body there
is there any body doing methylation analysis using epitect bisulfite kit from qiagen
actually I am facing the problem that after bisulfite treatment and purification, when i load the sample in 1.5% agarose no band is visible.
I started with 2ug of DNA and eluted in 30ul of EB buffer and loaded 6ul on agarose gel
can anybody help me to troubleshoot the problem??
After bisulfite modification and purification, you may not necessarily see the DNA on a agarose gel because there is huge DNA loss and degradation during these process despite the epitect kit is among the best for DNA recovery. Just go ahead with PCR to see if you can amplify anything from the treated samples.
I've been using the Epitect kit lately and I think its great. I haven't run any of the final product on a gel, but I have got most of my PCR's to work with some tweaking. I also used 2ug and eluted in 3ul elution buffer. I also use 1.5ul of this in a 25ul PCR reaction. The sooner Bisulphite conversion kits come with positive control primers the better in my opinion!
Good luck with the PCR's.
Davo, the HGS MEthyleasy Kits come with just that! positivie bisulfite converted DNA control DNA for conversion and primers for bisulfite PCR.
Dang, guess who just bought a Qiagen kit?! It's still a good kit though, but +ve control DNA and primers sure is an advantage.
I´m using also the Qiagen EpiTect and i never had Problems.
For Elution i usually elute twice with 20µl (starting from 1-2µg gDNA) and this encreases the yield.
From this i take 1µl for my PCR´s and i works always great.
I only had some problems when i start with less dann 500ng DNA so i try to avoid this.
You are probably using well designed primers too. How big are your PCR products? (Sounds like a rude question ) The largest I have to work is close to 700bp but the 400-500's work better.
I have framgents between 600 and 300, i must say
i never had to amplify a longer fragment :-)
I think, beside the quality of the Bisulfite DNA you need also a realy good PCR set up, this includes also
the Polymerases. I tried different Polymerases and with one i got no product while the other one works great.
Care to share the name of your polymerase? I've been using Plat Taq from Invitrogen. Some of my PCR's are brilliant and some are ok. Some don't work at all but I am not blaming the polymerase for this.
Lower the extension temperature for your PCRs to 64-66 and extend the time slightly. Very high AT regions will not amplify with 72C extension temperatures. These are quite common in bisulfite converted samples.