4 replies to this topic

[rmbio]

Hi

I have been trying to optimize sonication of the chromatin from rat brain for ChIP. Please find below the gel image of the decrosslinked, proteinase K treated and phenol washed DNA. Sonication was done with Bioruptor (1) 5 (2) 10 (3) 15 and (4) 20 cycles of 30 sec on/off at high power. Does any of these chromatin samples appears to be of the quality of being taken ahead for immunoprecipitation? I can provide details of steps performed after tissue harvest and before sonication.
Thank you in anticipation

Edited by rmbio, 03 October 2012 - 05:22 AM.

[pcrman]

Lane 3 and 4 have a nice distribution of the fragments, but seem a bit over sonicated. Line 2 is not enough.

[ChIP Kick]

Hi,

Is your problem solved? I have some problems with my ChIP assay. Hope you can give me some suggestions.

I sheared some formaldehyde cross-linked tissues samples. Then, treated with RNase A at 37C for 30min. After adding protinase K, reversed crosslinking at 62C for 2hours. Then run a 1% agarose gel with 85voltage.

Here are the problems. I do not know why there are red lines(in attached file) in my gel when running? In gel images, why I do not have smeared DNA?

My sonicator is Qsonics Q125. Sonication with 30% power, 15sec on, 45sec off, 10cycyles.

I spend several weeks to do ChIP but still cannot get good DNA fragments. Frustrate Now!

Attached Thumbnails

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[rmbio]

I did not do any experiment after above attempt.
What's the marker size in your case?  I have consistently observed that if u load the sonicated samples directly (without phenol purifyling and having 1% SDS coming from lysis buffer in my case) onto the agarose gel, you get some weared pattern. I am sure you have done the same. So my suggesiton is that after RNase-proteinaseK incubation give phenol-chloroform treatment followed by alcohol (isopropanol or ethanol) precipitation and than load the samples on gel for analyzing the size.

[ChIP Kick]

Hi rmbio, the marker in the fist lane is 1Kb Plus DNA ladder and in the last lane is 100bp DNA ladder. I will try the phenol-chloroform treatment. Thank you very much for your suggestion!