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ChIP purification method


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7 replies to this topic

#1 cellculturenovis

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Posted 27 July 2010 - 06:47 AM

Hi everyone!
Has anyone had problems using column based purification in their ChIP assay? I have DNA before purification but obviously it shows contamination and then after purification and treatment with RNase the DNA is gone? I think it may be blocking up the columns. Has anyone tried phenol purification for ChIP?

#2 biznatch

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Posted 27 July 2010 - 12:07 PM

Do you use PK (protein kinase) as well? We treat with RNase then PK then use the Qiagen PCR Purification columns, and don't have any problems. I can give more details if you'd like :) Someone in my lab tried phenol a few times, but if I remember correctly, they got a lower yield and it was a bit less consistent than using columns.

Edited by biznatch, 27 July 2010 - 12:11 PM.


#3 cellculturenovis

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Posted 28 July 2010 - 01:31 AM

Hi
I am still designing the protocol as no one in my lab has ever done ChIP before. I am still optimising the sonication time but to do this i need to purify it and run it on a gel to check the band size. At the moment after lysis and sonication i am treating the samples with RNase A and proteinase K and heating the sample to 65 degrees C overnight to reverse crosslinks and then purifying using a Qiagen pcr purification kit.
How many cells are you using per ChIP and how long do you sonicate for? It could be that my fragments are still too big and are blocking the column?
Could you possibly send me the protocol?

#4 biznatch

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Posted 28 July 2010 - 07:16 AM

We use a BioRuptor to sonicate part of a mouse brain, not sure how many cells it is exactly. The protocol we use is basically the EZ ChIP protocol (Millipore), which is available online, except we make all the reagents ourselves or buy them separately, because it's cheaper. The protocol discusses how many cells to use, what volumes, and suggestions on optimizing sonication, which is advice that can be applied to any ChIP protocol.

To test sonication, you should be heating (in salt solution, NaCl) overnight first, then treating with RNase then PK. You don't have to purify, just take 20-30uL after PK treatment, add loading buffer and run on a gel.

The PCR purification kit purifies up to 10kb, it's unlikely that your fragments are bigger then that even after a little sonication. When you run the gel do you see anything stuck in the wells?

Edited by biznatch, 28 July 2010 - 07:18 AM.


#5 cellculturenovis

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Posted 28 July 2010 - 07:33 AM

Thanks for the help.
No there is nothing on the gel or stuck in the wells.

#6 Lapsang

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Posted 28 July 2010 - 07:50 AM

I've never used columns for ChIP (the guy I work for at the moment has something against them).

After proteinase K I've always done phenol/chloroform followed by ethanol precipitation and it's been fine for me.

#7 biznatch

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Posted 28 July 2010 - 08:08 AM

I used to phenol/chloroform extract for checking sonication based on a protocol from someone else in my lab, which was based on the EZ ChIP protocol. Then I got myself a copy of the original EZ ChIP protocol and saw they don't have a purification step, you can just run some on the gel after PK. And the other person in my lab said they didn't know where their purification step came from they got their protocol from someone else :rolleyes: . So I tried without purification and it worked just fine. The moral of the story is, don't just follow someone else's protocol blindly there might be a better or faster way to do things :)

At the end of the ChIP protocol when it comes time to isolate the final DNA for PCR, we do however use columns to purify.

Edited by biznatch, 28 July 2010 - 08:09 AM.


#8 cellculturenovis

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Posted 29 July 2010 - 01:12 AM

Thanks for the help i will give it a go!




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