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Quantify DNA before ChIP PCR

ChIP QPCR Enrichment

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3 replies to this topic

#1 Mindbomb

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Posted 23 August 2012 - 04:30 AM

I did a ChIP experiment for a transcription factor. Just after the chromatin shearing I separated 5% as input. Now as everything is finished I would do the QPCR. I'd like to find out the % from input. Do I need to quantify the ChIP and input samples before PCR and standardize them to the same level? Thanks!

#2 chabraha

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Posted 27 August 2012 - 09:10 AM

Short awnser is no.
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#3 Mindbomb

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Posted 31 August 2012 - 08:34 AM

Thanks! I would like to compare protein binding to DNA between two samples. Is the best way to just use the formula 2^(Ct background-Ct binding site) in order to find the fold change, in which case the Ct background is measured based on the same IP sample? I was worried about the DNA quantity as differences there can lead to different Ct values between samples. Is it necessary to incorporate the input sample somehow in the formula?

#4 chabraha

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Posted 03 September 2012 - 09:49 AM

the delta-delta method is fine for ChIP............the way i do it is by calculating a %Input value for each target by using the input Ct value...........SA Biosciences has a good description on the math involved for this step. Basically you will have a % Input value that tells you how much of the total avaliable target region you were able to IP..........I calculate a %Input for both a specific IP (TF of interest) and a non-specific IP (IgG or isotype) I subtract the non-specific from the specific and now you have an IgG corrected %Input. Look at this value at a region known to bind your TF of intererst and a region known NOT to bind your TF of interest..............the differences there should be significant in order to call it a successful ChIP.
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