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Bisulfite treatment


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7 replies to this topic

#1 criscastells

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Posted 11 May 2010 - 12:34 PM

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

#2 criscastells

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Posted 11 May 2010 - 12:39 PM

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

#3 epicrazy

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Posted 12 May 2010 - 02:26 AM

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

#4 criscastells

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Posted 13 May 2010 - 05:22 AM

Hi epicrazy.

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

#5 methylnick

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Posted 18 May 2010 - 05:35 PM

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

#6 epicrazy

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Posted 19 May 2010 - 06:11 AM

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...

#7 epimaster

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Posted 27 May 2010 - 09:59 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

#8 criscastells

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Posted 01 June 2010 - 07:18 AM

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




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