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EZ DNA Methylation-Lightning Kit Incomplete Conversion‏

conversion bisulfite incomplete

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#1 Lj Lee

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Posted 08 September 2014 - 05:35 PM

Hi all,

 
Good day. 
 
I purchased a EZ DNA Methylation-Lightning Kit and I did my conversion following the exact conditions stated in protocol with 500 ng of DNA input. However, I found some incomplete conversion in my converted DNA. Is there any way that I can optimizes some parameters to improve the conversion? Like increase the denaturation time at 98C or prolong the incubation period at 54C? Or is there any other ways I can improve my conversion?
 
Do give me some suggestions. Thank you very much. rolleyes.gif 


#2 phage434

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Posted 08 September 2014 - 06:55 PM

How are you assessing your incomplete conversion? The only time I've had problems was with converting small circular plasmids. For those, I had to linearize them, and then had no issues.



#3 Lj Lee

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Posted 08 September 2014 - 10:27 PM

How are you assessing your incomplete conversion? The only time I've had problems was with converting small circular plasmids. For those, I had to linearize them, and then had no issues.

 

Hi phage434,

 

I assessed the conversion by using two set of housekeeping actin primers, no CpG sequence are present in the target thus the cytosines are always unmethylated. While another set of primers target the exact sequence but was designed to target unconverted gDNA. I run my converted DNA using both set of primers and I get bands in both. So this indicated incomplete conversion.  



#4 phage434

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Posted 09 September 2014 - 05:59 AM

Is this an real time PCR reaction, or an end-point reaction? I would not be at all surprised if sufficient residual unconverted DNA was present to template an end-point PCR reaction. In general, I would suggest sequencing and looking at the peak heights of your electropherogram. Not only could conversion be incomplete, but methylation of the sites is almost certainly not complete within the cells.



#5 Lj Lee

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Posted 09 September 2014 - 05:42 PM

Is this an real time PCR reaction, or an end-point reaction? I would not be at all surprised if sufficient residual unconverted DNA was present to template an end-point PCR reaction. In general, I would suggest sequencing and looking at the peak heights of your electropherogram. Not only could conversion be incomplete, but methylation of the sites is almost certainly not complete within the cells.

Hi phage434,

 

It's an end-point PCR. Thanks for enlightening me, is it better if i switch it to real-time PCR? Actually I plan to do qMSP but I was thinking if the conversion is incomplete it may give false methlyation signal when I run it on qPCR. I understand that the methylation site within cells is certainly not complete therefore I have to eliminate the possibility of incomplete bisulfite conversion.

 

Thanks.  



#6 phage434

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Posted 09 September 2014 - 06:11 PM

You could do a real time PCR reaction, which, with the correct primers, would tell you the relative amounts of methylated vs. unmethylated sites at a single locus. Better would be to amplify a region of interest with PCR and then sequence to find methylated sites.







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