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Is gDNA digestion before bisulfite conversion important?


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#1 methdetector

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Posted 15 July 2009 - 12:04 PM

Hello,

I am not sure if I should digest the genomic DNA before I treat it with bisulfite ( I am using EZ DNA methylation-direct kit). I have been told that very long DNA strands might come back together and do not allow the conversion process to be completed. Is this true?

If so, what are the 'general' enzymes recommended for gDNA digestion?
Any suggestion would be greatly appreciated.

Thanks :D

#2 MoB

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Posted 16 July 2009 - 04:34 AM

I am not sure if I should digest the genomic DNA before I treat it with bisulfite ( I am using EZ DNA methylation-direct kit). I have been told that very long DNA strands might come back together and do not allow the conversion process to be completed. Is this true?


Disgestion is not needed when using the Zymo kit. It performs excellent even on high-molecular weight DNA.

If so, what are the 'general' enzymes recommended for gDNA digestion?
Any suggestion would be greatly appreciated.


Any enzyme which does not cut in your region of interest ;-)

Best,

MoB

#3 methdetector

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Posted 24 July 2009 - 12:01 PM

Thank you very much!! :wacko:
It really helps


I am not sure if I should digest the genomic DNA before I treat it with bisulfite ( I am using EZ DNA methylation-direct kit). I have been told that very long DNA strands might come back together and do not allow the conversion process to be completed. Is this true?


Disgestion is not needed when using the Zymo kit. It performs excellent even on high-molecular weight DNA.

If so, what are the 'general' enzymes recommended for gDNA digestion?
Any suggestion would be greatly appreciated.


Any enzyme which does not cut in your region of interest ;-)

Best,

MoB



#4 et2b

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Posted 12 August 2009 - 12:07 AM

Hey,

I always get worse results when working with lower mol. gDNA.... Or Am I just a silly person ?
EZ DNA methylation works like a charm with high mol. gDNA. Got my latest ABI traces back >99.8% conversion...
got to love it...
(And I AM loving it B) )

Bye

#5 methylnick

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Posted 13 August 2009 - 01:40 PM

I always get worse results when working with lower mol. gDNA.... Or Am I just a silly person ?


You are not :lol: , low mol gDNA gets chewed up by the conversion reaction, it is a degradative process. If you are working with low mol gDNA design your assays a little shorter than normal.

We work with FFPE samples and they are a challenge to work with to say the least! ;)

#6 et2b

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Posted 14 August 2009 - 12:45 AM

I always get worse results when working with lower mol. gDNA.... Or Am I just a silly person ?


You are not :) , low mol gDNA gets chewed up by the conversion reaction, it is a degradative process. If you are working with low mol gDNA design your assays a little shorter than normal.

We work with FFPE samples and they are a challenge to work with to say the least! ;)


Hi Methylnick, thanks for the ABI tips, and FFPE and bisulfite.... I feel your pain!!!

good luck, many thanks




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