what to resuspend the DNA pellet after DNA isolation step? - (Jan/02/2008 )

in the ChIP assay, i used TE buffer to resolve ChIP product, but got no result, this is the fifth ChIP ,i was very confused
have i used the wrong buffer?
help me !

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The EDTA in the TE might be interfering with your PCR (though it's such a small amount). You could always try just using 10mM tris (preferably pH 8) or DI water (but keep in mind that DI water is often slightly acidic and DNA is more stable in a basic solution).

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In my opinion, it does not vary too much between TE or DI water-dissoved DNA. I've tried both. If you got no DNA band in your PCR, I am afraid you should:

1> check the lysate (or input signal);
2> change another set of primers.

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thanks !
i will use 10nM Tris in my experiment. i checked the bands, the input control had bands ,but i got no band in IP sample(added antibody)

maybe these buffer in ChIP didn't work?
or other reason,(antibody or the beads)?

can anyone tell me the most possible cause?

thanks!



protocol:http://openwetware.org/wiki/ChIP

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There are quite a number of things that can go wrong in ChIP. Good positive controls will help you pinpoint what's going wrong.

1) Do you have a few positive control primers (primers for a region where you know your factor of interest binds)?

2) If you don't get amplification in your IPs with positive control primers, then you should check to make sure ChIP is working for you. Try ChIP with an antibody which many people have had success with and for which the pattern of its binding is well known (like to H3 or Pol II or H3K4me3).

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