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help on % input calculation please!!


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#1 chipy

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Posted 14 December 2011 - 09:29 AM

Hi,

Here's my problem: I just performed a ChIP experiment in which

1. After cross-linking and sonication I took 5 ug of DNA for input and IPs samples.
2. After, IP, cross-linking reversal and DNA purification I eluted DNA on 30 ul (all samples including input).
3. I performed a qPCR raction using 1 ul of input and 3 ul of IPs samples.

Questions are:
1. How do I calculate the % input?
2. Is it absolutelty necessary to tun a standard curve whith input dilutions or can I just aplly the 3,3 Ct differences over 10 fold dilutions??!


Thanks in advance I'm stuck on this one for so long now!!

#2 chabraha

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Posted 14 December 2011 - 03:29 PM

you need to calculate your %Inputs by factoring in the dilution factor with the difference between your Cts..........in Real Time PCR ~3.32 cycles is a 10 fold difference. For example, if you pulled 5ug of DNA for your input and you ChIPed 500ug thats a 100 fold dilution (1%).........let say for simplicity also that you added equal amounts of Input and ChIPed DNA into your PCR reaction..........you get a Ct of 24 for your Input and a Ct of 25 for your ChIPed DNA..........you would first calcuate your percent input by (2^-(25-(24-6.64)))*100.................you use 6.64 in this instance because 6.64 converts your 100 fold dilution into cycles.........................hope this helps.
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