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how to do % input calculations from Ct values

ChiP qPCR

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#1 Sandeep Gupta

Sandeep Gupta

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Posted 19 July 2017 - 12:33 AM

Hi All, 

I am currently standardizing the ChiP protocol in my lab and very naive in the technique. I performed a ChiP protocol with a protein specific antibody and Igg control and after precipitation, I resuspended the pellet as well as 10% input sample in 100 microliters of water. I do not have bio analyzer so wasn't sure about the concentration of the precipitated DNA, but could get the concentration for 10% input sample. I diluted the 10% input to 1% input and used 4 microliters of input as well as precipitated DNA in the qPCR reaction. I used primer set-1 which should recognize the bound region versus the primer set- which lies 2kb outside of my protein of interest bound region. I can normalize it by Igg ct values, but don't understand how I can use the input Ct values. The input Ct values will always be much lower than the precipitated one so a simple normalization is not working. Can anyone suggest how to calculate the % input in this case? 

Here are the Ct values: 

Wt-1%----33.8 Wt-Igg----40 Wt-SM1--39.3 WT-sm5--37.6  
 

 

Thank you so much in advance!! 

 

Best 

Sandeep 







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