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Bisulfite conversion kits


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6 replies to this topic

#1 criscastells

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Posted 01 October 2010 - 03:25 AM

Hi everyone,

I have been working with methylation studies but I can not obtain good results. I have used a bisulfite conversion kit from Qiagen, another for Ecogen and right now Invitrogen gave me a sample of the menthylcode bisulfite conversion kit.

I would like to open a discusion between those people that has used these kits or another ones to say what they think about (it works, it not, it degrades the DNA, it is needed highly purified DNA,...)
Everything you coment would be a help for me to decide what to do.

Thanks you very much

CRIS

#2 methylnick

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Posted 01 October 2010 - 04:47 AM

can we make a start by asking what sample type you have, this has major bearing as to why you have not been successful with your conversions.

How were your DNA's prepared? what amount did you use for conversion and what controls you performed to assess that your conversion has failed?


Nick

#3 epigenius

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Posted 01 October 2010 - 06:12 AM

Qiagen is a pretty robust protocol in my opinion, so Methylnick has a good point.

#4 criscastells

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Posted 04 October 2010 - 01:26 AM

I work with in vitro three dimensional cultures using as a culture control 2D samples. The cells are fibroblasts and I have used different amounts of DNA to do the bisulfite conversion (between 100 and 500 ng). I isolate the DNA using a QIAamp column obtaining a good A260/280 ratio and an acceptable A260/230 ratio. The genomic DNA extracted looks OK in an agarose gel.
I do not say that the conversion has failed, but the most part of DNA is degraded obtaining only 1/3 or less quantity of the DNA used for the conversion.

Probably I am doing something in the incorrect way but I can not found what it is. I will be really greatful if you give me some advice.

Thanks

#5 methylnick

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Posted 05 October 2010 - 05:03 PM

Bisulphite actually degrades your DNA so it is expected you will get loss of DNA from the process, the only kit i know of so far that doesn't do this is the Human Genetic Signatures Kit, MethylEasy, although I think Qiagen claims the same now.

The next question for you now is, how are you determining your bisulphite conversion has worked or not? Nanodrop? PCR? or other means?

#6 criscastells

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Posted 06 October 2010 - 12:59 AM

I know that the bisulfite degrades DNA, but what I did not expect was to obtain only a 40% of recovery while the kit talks about an 80%. I used both PCR and Nanodrop for determine that the conversion works or not.

#7 epigenius

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Posted 06 October 2010 - 07:01 AM

When you say recovery, as opposed to conversion, are you taking the spin column filtering into consideration? You will lose some of the recovery after passing through the spin column's filter membrane no matter what, even if you get 100% conversion, which is typical regardless of which kit you use.




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