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Old 03-22-2014, 01:07 AM   #1
korchid
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Location: melbourne

Join Date: Oct 2013
Posts: 1
Question Library preparation for negative control sample?

Hello,

I'm wondering if anyone has experience with making libraries with your negative control ChIP sample? I am trying to pull down a particular factor in the wild type cells and also performing the same experiment with cells not expressing that factor to serve as the negative control as the antibody is not that specific, and there is a certain degree of non-specific binding judged from my previous sequencing result.

Now I'm trying to repeat this experiment and make libraries for both of these two samples again. My question is during the PCR enrichment step, if I PCR amplify my negative sample additional cylces to have enough material for making the library, will that reduece my signal-noise ratio? My concern is that the antibody will pull down something from the genome even in the cells not expressing the factor, and if it's over amplified to a degree that the signal is similar to that from my actual ChIP sampe from the wild type cells, does that mean potentially I will lose some real binding site, as there is amplicon showing up in the negative sample as well...

I don't know if this is making sense, but if you have any experience or suggestion, that would be highly appreciated..

Thanks!!
Korchid
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