Phenol/chloroform vs Column purification for ChIP DNA - (Nov/24/2008 )

Hello all, after receiving such helpful advice on a previous post I will continue to extract knowledge out of you people:

Want to know pros/cons versus the two techniques to purify my chromatin at the end of a ChIP protocol.

Our lab has the Qiagen kit which I know how to use and seems the easiest, problem is I am starting with about 500 microliters of eluted chromatin (which is then diluted 5x) and the columns only hold 800 microliters. I could use multiple columns but then I end up with very diluted DNA for qPCR. This seems like a problem.

As am new to the world of science have yet to phenol/chloroform anything although have done just chloroform extraction during genotyping. What exactly do I need for this procedure? I assume after precipitation I could then resuspend in any volume I wish therefore fixing the above problem, correct?

Appreciate any help,
Stephen

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If you are going to use columns I would suggest using Qiagen mini-elute columns, or something similar, since they have lower retention than the normal columns (which is important if you are dealing with small amounts of DNA like when doing ChIP).

If you are just getting started with ChIP I'll plug the method we developed. It's based on the fact that much of the cleanup procedures that people do at the end of ChIP are unnecessary and too time consuming. The method is very fast and very simple:

Essentially, after the last wash of the IP, add 100µl of 10% chelex resin (BioRad) and 20µg proteinase K directly to your beads. Incubate at 50-55C for 15-30min and then incubate in boiling water for 10min (don't worry, the DNA is safe in chelex). Spin down at 4C (to cool and get all of the condensation to the bottom) and transfer the supernatant to a new tube. Wash the beads/slurry mix with 100µl of water, spin down, and pool the supe with the previous supe. The pooled supe is ready for PCR. This method works great if you're doing PCR but don't need to do ligate adapters to the end of your ChIPed DNA for whole genome amplification, since you will end up with single stranded DNA after the boiling step.

Also, if you are interested, we have a protocol for ChIP in 96 well plates that takes about 4 hours to do 96 ChIPs and uses about 10-20 fold less chromatin per ChIP. Let me know and I'll send you a protocol.

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Thanks KPDE,

Appreciate the protocol, let me slog through the traditional before I am confident enough to change it up.

Have now figured out phenol/chloro extraction is no big deal, though has the potential to be a bit hazardous to ones health. Is there any way why I couldn't just precipitate the combined eluates of the columns in ethanol. That way I could then resuspend in whatever volume I desired, correct?

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Hi Stephen,

Column purification (for example use QIAquick PCR Purification Kit) saves you a lot of pain compared to phenol/chloro extraction. I know the sample volume is big (500 ul + 3x PB buffer), but you can load repetitively the same sample to the same column after filtering out the previous load before going to the next step. You can buy extra PB buffer from Qiagen because you are going to use a lot of PB buffer.

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Hi Stephen,

We use a QIAGEN kit to purify our ChIP samples. We end up with 200ul after elution and then add 5 vol of buffer PB (instead of what is in the normal protocol). The vol is too large to fit in the column at once so we fill it, spin and then fill it again. No problems
I have tried phenol/chloroform extraction but I got ugly real time curves so never bothered to try it again. And not to mention the kits are easier!! Can you elute your IPs in a smaller volume than 500ul?
Clare

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Well that sounds great, did not know that, thank you.

So in your opinion is there any upside of using phenol/chloro for ChIP?

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I may be able to elute in smaller amounts, but if I didn't do you think a single column could handle 2.5ml of sample (500 microliters + x5 PB)?

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I think so. When I first started chipping, I tried all sorts of purification kits and back then I eluted in a larger volume. Just give it a try
Clare

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Sure thing.

One thing I'll mention though ... the only reason for the phenol:chloroform or column cleanups in the traditional ChIP (if you are only analyzing by PCR) is to get rid of the SDS and bicarbonate in the elution buffer, which if you didn't add it in the first place, you wouldn't need to cleanup.

The cleanup of your ChIPed DNA is unnecessary IF you don't elute with a buffer containing components that are inhibitory to PCR. So, you can elute using 25mM tris pH 10, 1mM EDTA and 20ug proteinase K (heat at 50-55C for 15 min then 100C for 15 min; the elution and reversal of crosslinking happens simultaneously) and your DNA is then PCR ready without any further steps.

I mention it here because, when you are just getting started with an assay, it should be the simplest version of the assay possible, so that if you have to diagnose problems, you have less steps to diagnose. And, if you start to ramp up the number of ChIPs you do (100-200 a day) you definitely want the simplest protocol possible.

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