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Amplicons detected for negative control sites (ChiP-qPCR)


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#1 vic17

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Posted 10 October 2012 - 09:58 AM

Hi, I am new to ChiP-seq and would like some help with negative control sites. I'm doing a H3K27ac ChiP. My negative control sites primers are amplifying and this resulted in Ct values of around 23-26 for the ChiPed sample and quite similar values for the input. The target region would yield Ct values around 21-23. The average fold change (target sites over negative control sites, both normalized to inputs) I often get is around 5-7. My questions :

1) what are the normal Ct values one would expect to see with negative site primers? I was expecting around 30-35 (or higher) since in theory they should not be amplifying anything?

2) what is the typical/acceptable fold change that would work well for the hi-seq?

Appreciate any help given. Thanks!

#2 trungbiotech

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Posted 27 November 2014 - 10:29 PM

I think as long as you have more then 5 fold difference between positive and negative regions, it should be ok






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