Chip Assay - PCR step (Feb/13/2008 )

hi guys
I am doing a chipassay and I am at the final step for PCR.

I want to compare my sample A with and without drug for their binding to a transcription factor T.

so I pulldown with T then follow the chip assay protocole.

I also did an input meaning no puldown (total lysate)

For the PCR step I included a PCR positive and negative control (plasmid with A and pcr with water).

waht other negative control should u use?

another question to be able to compare my pcr product i need an internal control. correct?
as an housekeeping gene to be able to compare. but I have no idea if the house keeping gene will be in my pull down.

any advice for this assay?

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I'd use the ORF of the gene you are looking at as a negative control. Then you have enhancer vs. ORF, which will give a nice graph, no?

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ok so for the PCR do you quantify the DNA before PCR to use the same amount or you considere that by pulling down the same amount it should be fine.

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I don't have an absolute quantification. I do a standard curve with a certain percentage of the input material I use for the ChIP, for each primer pair I use.
Then I calculate, how much is actually pulled down of the regulatory region vs. the ORF (which is not supposed to be bound).

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