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Typical ChIP yields


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#1 jamessmith01

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Posted 17 August 2010 - 08:11 AM

I am just wondering what kind of yields people are getting from their ChIPs.

I currently get around 4-5 ng of DNA from a H3ac ChIP, using 4 ug of chromatin and 2 ug of antibody. I'm using the Active Motif ChIP-IT express kit (magnetic beads).

If this is massively short of what others get, I might get some new antibody (it's supposed to be a good one, well published, but could I have knackered it with multiple freeze-thaws?). I'm also going to run a PolII ChIP to check what a good yield from this kit should look like.

Thanks.

#2 Mighty Mouse

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Posted 20 August 2010 - 01:44 PM

I am just wondering what kind of yields people are getting from their ChIPs.

I currently get around 4-5 ng of DNA from a H3ac ChIP, using 4 ug of chromatin and 2 ug of antibody. I'm using the Active Motif ChIP-IT express kit (magnetic beads).

If this is massively short of what others get, I might get some new antibody (it's supposed to be a good one, well published, but could I have knackered it with multiple freeze-thaws?). I'm also going to run a PolII ChIP to check what a good yield from this kit should look like.

Thanks.



I've never quantified my ChIPs that way; I usually figure out my "yield" by running qPCR and comparing to input, thereby getting a % input. For TFs I get % inputs around the 0.1 - 1.0 % range. For histone modifications I get something between 5-30% input. I'm not sure that quantifying the amount of DNA you've pulled down is a particularly good measure of how well your ChIP has worked; I would recommend doing PCR on a region that you know binds your factor and compare that to a negative control region (e.g, LINE1 or 28S rRNA etc.) and see if you have greater binding in your positive versus your negative control...

MM
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#3 jonny_boy

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Posted 30 September 2010 - 07:43 AM

Hi Mighty Mouse,

I have a quick question for you! I have been doing histone ChIP for a while now but have never had enrichment as high as you have stated. However, I am wondering whether this may be because I am classifying my 'input' in a different way to you? When you calculate percentage input do you do it based on the total amount of chromatin placed into the ChIP reaction or do you compare to a smaller aliquot (i.e if you precipitate 50 ul of sheared chromatin does your input sample also represent 50 ul or a smaller amount, say 5ul as suggested in Millipore's EZ-Magna ChIP kit)?

Thanks in advance for the response!

Jon

#4 Mighty Mouse

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Posted 30 September 2010 - 08:41 AM

Hi Mighty Mouse,

I have a quick question for you! I have been doing histone ChIP for a while now but have never had enrichment as high as you have stated. However, I am wondering whether this may be because I am classifying my 'input' in a different way to you? When you calculate percentage input do you do it based on the total amount of chromatin placed into the ChIP reaction or do you compare to a smaller aliquot (i.e if you precipitate 50 ul of sheared chromatin does your input sample also represent 50 ul or a smaller amount, say 5ul as suggested in Millipore's EZ-Magna ChIP kit)?

Thanks in advance for the response!

Jon


Hi Jon,

I usually pull out 1% input. So I'll have a 500uL volume for my chromatin/dilution buffer/antibody/PI mix, then I'll pull out 5uL as my input. Then when I do my calculation I'll do 0.01*2^dCt where dCt is the Ct difference between my input and my target. I use the 0.01 to "correct" for the use of 1% input for my final calculation. I like to use 1% input because typically I'm ChIPing for TFs and my qPCR curves for my input and factors are fairly close thereby minimizing any distortion due to primer in-efficiencies over long Ct ranges. I hope that's clear...

MM
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