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ChIP protocol


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#1 lsek

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Posted 01 June 2009 - 09:19 PM

Hi Everyone,

I need help. I working on ChIP and was wondering if anyone is doing ChIP with solutions prepared by themselves.
Could you help confirm if the components and its composition are correct:

ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl, pH 8.1)
ChIP dilution buffer (0.01%, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl)
Low salt immune complex [for wash step 1] (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 150 mM NaCl)
High salt immune complex [for wash step 2] (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 500 mM NaCl)
LiCl immune complex [for wash step 3] (0.25M LiCl, 1% IGEPAL Ca630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl, pH 8.1)
TE buffer (10 mM Tris-Cl, pH 8.1, 1 mM EDTA)

Elution buffer (1% SDS, 0.1M NaHCO3) prepared fresh by adding from stocks of 10% SDS and 1M NaHCO3.

Sorry for the trouble. I'm trying to troubleshoot my ChIP experiment which is not working. Ohya, do I need to add protease inhibitors for the washing steps? Thank you so much for the help.

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#2 madrius1

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Posted 02 June 2009 - 07:04 AM

I use homemade buffers, and my experiments work pretty well. Here, i'll compare to your buffers.

ChIP lysis : 1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1. Add 1mM PMSF and 1X protease inhibitor cocktail before use.
ChIP dilution buffer : 0.01% SDS, 1.1% Triton, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl. Store at 4C and add 1mM PMSF and 1X protease inhibitor cocktail to the required volume for the ChIP before use.
Wash 1 (TSE-150) : 0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl
Wash 2 (TSE-500) : 0.1% SDS, 1 % Triton, 2 mM EDTA, 20 mM Tris pH 8.1, 500 mM NaCl.
LiCl : 0.25 M LiCl, 1% NP40, 1% DOC, 1 mM EDTA, 10 mM Tris pH 8.1.

TE and Elution are the same. So your buffers are the same as mine!

And no, you don't need protease inhibitors in the washing steps.

#3 lsek

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Posted 04 June 2009 - 08:32 AM

Thank you so much! :o

Sorry for the late reply.

Cheers.

Edited by lsek, 04 June 2009 - 08:32 AM.

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