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Variable ChiP-qPCR even in the same lysate aliquots

ChiP ChiP qPCR

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#1 Sandeep Gupta

Sandeep Gupta

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Posted 15 September 2017 - 02:22 PM

Hi All, 

I am trying to standardize the ChiP protocol in my lab and so far just got the frustration out of it. I am chipping SMADs in mouse ESCs. My major problem is the reproducibility of the results. I get really predictable results one time but not with the different aliquot of the same lysate. I lyse cells in 1ml and aliquot it right away in 300microl amount. The overall chromatin concentration is really good (0.5 microg/miL of lysate) and I use 25 miG for 1 ChIP reaction (say Igg). After eluting the DNA from beads I treat it with RNAse A for 1 hour at 37 and Proteinase K for 2 hours at 65. I usually clear the DNA by Qiagen PCR cleanup columns but I always get DNA yield of 0.2 to 1 ng/miL which is so little. With this DNA i set up qPCR and every time I run it, it gives me completely different results. I am not able to reach to the conclusion that my ChiP is working. I eventually have to send chromatin for the sequencing but always get nervous since I am not sure if it is working. 

One time I did not incubate my samples in RNAse and Proteinase K and just cleaned up the DNA using Qiagen columns. This is the first time I got 10ng to 50 ng/miL DNA yield and my qPCR worked fantastically. I am not sure If Qiagen columns are sufficient to remove RNA and proteins in the sample and can I use this template for the sequencing. 

I would really appreciate if anyone has gone through the similar situation and any sort of advice of what I might be doing wrong. 

 

Thank you all in advance and eager to listen what you might have to say. 

 

P.S: the gel pic of chromatin shearing is attached here. Webp.net-compress-image.jpg

 

Best, 

Sandeep 







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