ChIP DNA concentration - (Apr/19/2007 )

hi everyone,

im new to chip'ing and have just a few questions...

how important is it to determine how much dna is in your chip sample? also is it normal to measure the concentration of the final dna samples before performing the pcr reactions? i havent been measuring the concentration and have just been using the same VOLUME of dna for every pcr reaction instead of determining an exact concentration. i have been advised to use the same amount of dna for every pcr reaction but im not sure this is correct. if i wanted to compare the level of interaction of 3 different proteins to 1 specific gene should i be using the same amount of dna?

any help would be appreciated as i really dont know which way is correct.

thanks.

--missh

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10ng of DNA is enough to conduct PCR. So, You don't condern about the concentration of DNA. In my case, I conducted ChIP with 3x10^5 cells and then used 1/10 diluted elution solution as template for PCR

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much of a muchness really.

We use the same amount of DNA for our PCR's with the assumption that in the enriched fraction, you would get more copies of you target gene than in the same amount of the input. This is particularly important if you are performing a WGA before array, or PCR.

Nick

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Normalization of the input DNA seems to be necessary for accurate quantitation of enrichment. But with that in mind, does the salmon sperm DNA also elute from the beads? If so, couldn't it potentially interfere with the quantitation simply on the basis of amount of "input" DNA? Or does it sort of act as an inert carrier if you assume that equal amounts of SS-DNA are present in each sample?

-jb

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jb,

salmon sperm DNA will co-elute, but because you have same amounts of it in both fractions, it becomes inert in your downstream PCR applications.

Cheers

Nick

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I agree, the amount of SS-DNA should be equivalent in all the samples. The question boils down to how much SS-DNA elutes compared to the actual ChIP'ped DNA. I assume that if SS-DNA is present in vast excess, the ChIP'ped DNA contributes only a negligible amount to the final DNA concentration, and what we're really quantitating is the relative elution/purification of DNA in the various samples and controlling for slight variations between them. Is this correct?

-jb

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Rather than worrying about quantitating your DNA after the ChIP wouldn't it be easier to normalize your input before ChIP.

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I definitely agree that immunoprecipitating from equal starting amounts is important, but the quantity of IP'ed DNA for each antibody will differ depending on the amount of antibody used, the affinity of the antibody for the its target, the relative abundance of the target epitope in the sample, etc.

I can see both sides of the argument, why some people would quantitate DNA post-ChIP and analyze equal amounts of immunoprecipitated DNA, while others use equal volumes of ChIP eluate for the PCR. My question is still whether the amount of SS-DNA eluted from the beads is generally in excess to the amount of immunoprecipitated DNA, in which case the point of quantitating the DNA after ChIP is to normalize for variation in elution between the samples.

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