DNA purification after sonication - (Aug/22/2008 )

HI everyone,
I am tring to establish ChIP in my lab, and now I have some problems.
After the sonication, I want to verify the size of fragment. Do anyone have a protocol for the DNA purification by chloroform/phenol after sonication? And do you reverse-crosslink before going into DNA purification?
Sorry for these questions which might be idiot for you. I really need some helps.
Thanks!

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Seems there's no need to purifiy the sonicated DNA by phenol chloroform, but you can centrifuge your sample in 14,000rpm 10min 4C to remove cell debris

I also wonder if de-crosslinking is necessary

Affymetrix suggest this:

1. Add 100ul 10mM Tris (pH8) to 100ul sonicated sample
2. Add 2ul Proteinase K (20mg/ml)
3. 42C 2hr, then 65C 6hr (or overnight)
4. Clean up with Affymetrix cDNA cleanup column, elute with 20ul
5. Load 100-500ng of sheared DNA to an agarose gel

It seems very troublesome, and some ppl told me that it's not necessary, without de-crosslinking, you will see the same result

I wonder if anyone ever ran a gel without de-crosslinking too



JIRO

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Thanks for your answer, it's very helpful!
Just two more questions: 1. we have PCR purification kit from qiagen, do you think it works as your kit?
2. for the gel migration, I do it just as I do after a classic PCR, it's OK?

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1. Haven't tried but don't see why it wouldn't work
2. Yes, just regular 1% agarose gel in TAE, 1hr 100V

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Hi there

We do chips every week and find the easiest and quickest way to purify our samples is with a Roche column (high pure PCR I think).
It saves a lot of time!
Clare

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Hi,
In fact we don't have a kit to purify the DNA fragment．
I ordered one, but I must wait some time for its arrive. When I　ａｍ　ｗａｉｔｉｎｇ　ｆｏｒ　ｔｈｅ　ｋｉｔ　Ｉ would like to try the old fashion DNA purification.
Does someone have a detailed protocol?
Thanks

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Reversal of crosslinking is very important for looking at your fragment sizes on agarose. You will mostly get high molecular weight smears (sometimes the DNA will remain in the well) unless you reverse the crosslinks.

I do a huge volume of ChIPs every week so we have a quick way (~35min) to make input DNA for running on the gel and which is also PCR ready (as long as you don't have SDS is your sonication buffer above 0.1%). Add your chromatin (10ul or so depending on the concentration) to 100ul of 25mM Tris (pH 9.8 or above; yes I know it's out of the buffer range) with 1mM EDTA. Add 20ug of proteinase K and incubate at 55C for 15min. Then heat at 100C for 15min to reverse crosslinks (this is important for running on the gel; and yes your DNA will be fine since it's at a high pH). Then centrifuge at 4C to cool the DNA and run on a 1% agarose gel. The two most important things are to make sure that the 100C incubation is 95C or above and that the pH of the tris buffer is above 9.8 (if you make the buffer with tris base the pH, unadjusted, will usually be about 10 which is perfect).

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It will be very useful to decide whether the size of sonciated DNA fragment is among a appropriate range before addition of antidogy. If more volume of chormatin was used, should the amount of 25 mM Tris be increased proportionally? such as 20 ul of chromatin need 200 ul of 25 mM Tris.

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BTW, I read a protocol for fast ChIP published in "Nature Protocl" (Citation: Vol. 1 No.1,179-185,2006). Chelex 100 and boiling was done to reverse crosslink. But I don't have a try yet.

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Thanks, I will try this quick method.
Still have one question, after the PK digestion and the reverse crosslink, a centrifuge is enough to get DNA? I don't need to precipitate it?
Yeah it's true for running a gel I don't need high-purified DNA

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