Bisulfite treatment - (May/11/2010 )

Hi everyone,

I am trying to set up a protocol for methylation studies in our lab. I obtain genomic DNA and use 500microL for the bisulfite treatment. I know that this treatment degrades the most part of DNA but when I try to perform a PCR program I obtain a smear in the gel.

Anyone can give me any advice? I think that the problem is the DNA treated but it is possible that the problem is the PCR program.

Thanks

Sorry, I don't know it it could be important but I have use two different kits for the bisulfite treatment: EpiTect from Qiagen and EZ from Ecogen

Did you say you use 500ul of DNA? what is your bisulphite set up?

Hi epicrazy.

Sorry I do not use 500ug! I use 500ng!!!!! It was a typographical error!!!

the problem mainly stems to poor primer design and pcr conditions.

have your primers worked on bisulfite DNA in the past?

I am not familiar with the kits you have used, do they have control primers you could test your DNA upon?

The kit we use does, from Human Genetic Signatures

nick

Try scaling up your input DNA to 2ug, and run a PCR for 40 cycles... if you get a band or non specific, you can then optimise it...

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

Hi nick,

Thanks you very much for your advice. The primers I use are from a paper and I really can not reproduce the experiment. I tried to study these primers using bioinformatic tools but till some days ago I had problems to find the promoter sequences. Now I have used blast and I obtain no fragment result for these primers, so I think that these primers are not correctly designed and I will try to synthesize my own primers.

I wanted to ask you what do you think about to sinthesize my own primers considering that the promoter sequence is 500bp upstream of my gene.

Thanks again

CRIS