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Input too low in ChIP. Need some help please.


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#1 Epigene1

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Posted 09 August 2011 - 11:34 PM

Hello everyone!
Im new in the blog. I have been doing chip assays to look at histone modifications. I always have a problem with the concentration of my products. Everytime I measure the INPUT it is very low ( I have managed to get 20 ng the maximum) but when I run it on a gel it does not show any band. I use QIAGEN PCR purification kit to purify, I have also tried phenol/chloroform extraction but same results.

Anyone has any suggestion at all? I would appreciate it!

Many thanks for reading this!

#2 Muhammad Umer

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Posted 11 September 2011 - 12:35 PM

Hi
I guess you always have a control over input quantity. I have never carried out ChIP but what I know is, that you sonicate the samples and then take aside some amount of this sonicated sample as input. So if you start with a definite quantity of DNA, input must also be defined. Say for example you started with 2ug DNA in 200 ul (10 ng/ul) and then after sonication you take aside 20 ul as input and use remaining 180 ul for ChIP, the total DNA in your input must be 200 ng, which is more than sufficient.
As for being invisible on gel, 20ng is a low quantity I guess and you can never see it on gel




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