input problem from ChIP prep - (Apr/04/2008 )

Hi, everyone:

I have a problem for tinput from ChIP prep. I include the same amont of chromatin as I used fot my real IP samples to start reverse crosslink, then DNAase.proteinase K treatment. Finally I use phenol/chrom extraction and 100% ethanol with gycogen to precipetate DNA. When I apply those samples for Q-PCR, my ChIP samples look pretty fine, I got nice signal. But for my Input, there is almost nothing there, even the baseline on PCR amplifing profile is very high. I donot know what is the problem. Because after precipetation, I saw a big pellet in my input which I assume they are DNA. I throught it might be too concentrate in my input, and then I diluted down to 1 in 100 to set up Q-PCR. I still did not get any signal. what is my problem ? Do I need to use column instead of ethanol?

thanks advance for any help and suggestion.

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DNase Do you mean RNase?

Wow, that' s a lot. I took 10% of the sample as input and carry on immunoprecipitation with the remained sample. Why do you need so much material for the input?


That is most probably glycogen.

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But why my IP sample (the positive control which I know binds to the intersted region) works for the Q-PCR? I treated them the same way as my input during the ethanol pp.

thanks for your help, zek

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Do you also do normal PCR?

Have you determined DNA concentration in your input samples (maybe it won`t be so accurate but if you have a NanoDrop in the institute, you could try).

I really cannot think of anything else, sorry.

Good luck

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I am using Real time PCR and I did not try normal PCR yet for my input sample.
Yes, Zek, I do determine my DNA C before I go for the ChIP. Actually what I did was after finishing ChIP sample prep process (crosslink, lysis, sonication and pre-clear ), I frozen my lysate at -80 and take a small aount to reverse-cross link and determine DNA concentration by OD260. Then I will start my ChIP with 25ug chromatin per sample.

what is going on with my input

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I wish, I know the answer

I first do the normal PCR and then the real-time. The normal PCR is cheaper to check whether ChIP has worked or not.

You don't necessarily have to use columns for purification. Ethanol works for your samples, right??

Nowadays, I have the same problem in opposite way (no signal in samples, but beautiful inputs). After changing everything, I suggest our formaldeyde is the enemy. We have ordered a new bottle which will be aliquoted and used only for ChIP. If that does not work, I am out of suggestions.

Cheers

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