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Doubts Regarding Plasmid ChIP....

Transient ChIP Episomal DNA ChIP plasmid ChIP

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#1 chadi

chadi

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Posted 12 December 2013 - 01:41 AM

Dear all,

 

I have been trying recently to perform ChIP on transfected cells to find out if certain transcription factors and histones can bind to the transfected plasmid. I have always ended up having a huge amount of plasmid DNA and low amount of endogenous DNA thus rendering statistical analysis impossible. I have tried transfecting with as low as 1 microgram of plasmid and I still get the same problem.

 

Another problem that I have faced is that I am unable to find a negative control within the plasmid backbone. It is like whatever primer (specific to plasmid) that I use for qPCR, shows low cT and enrichment as if the transcription factor and the histone marker bind to the whole plasmid.

 

I have tried checking many references but I found out that the authors only use one primer pair specific to the plasmid and compare that with a negative control from endogenous DNA to do the statistical analysis. Is that a reliable way? (Like that I can safely assume that my experiments worked)

 

Is there anybody with experience on plasmid-ChIP who can help? Any reference that can be useful? Is plasmid-ChIP as a whole a reliable technique? Is there a special way of shearing that can help to enhance the results?

 

Thank you all.

 

With regards,

 

Chadi






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