CHIP - DNA concentration after sonication - (Apr/14/2009 )

Hello !

Can anybody tell me the concentation of the DNA after the sonication step?
How much DNA do you use when you incubate with the antibody. And how much antibody ?

Thanks in advance

rick2000 on Apr 14 2009, 09:28 AM said:

This is no easy question(s) to answer! The answers would all depend on how many cells you were using per ChIP, what kind of cells etc etc. You're going to have to be a bit more specific if you want a more detailed answer (from me anyway!)

Clare

My starting material was 1 gr of plant tissue. Do you take into consideration the starting material or the quantity of the DNA after the sonication step?

rick2000 on Apr 15 2009, 09:50 AM said:

From what I've heard people either use amounts of DNA or number of cells used. I go on number of cells personally... I think it's a bit tricky to quantify exactly how much DNA you've got after sonication because of all the associated proteins etc etc. Also, I don't want to waste any of it! (we use low cell numbers in our ChIPs)

Clare

Personally, I also use number of cells rather than fiddeling around with quantitating / adjusting the DNA after sonication. I recently did a sonication time course and was measuring the concentration of the (de-crosslinked / phenol-purified) Input material, and if that reflects a constant for the number of cells I typically use, I end up with around 25-30ug DNA per IP (with 20ul anti-mouse Dynabeads) - amount of antibody will greatly depend on the antibody itself, I usually use between 1ug up to 5ug....

Clare on Apr 16 2009, 03:32 AM said:

Yeah, quantitating the DNA after shearing can be next to impossible because of interference, not only by proteins but also by the protease inhibitors (and phosphatase inhibitors if they are used).

I do quantify my chromatin by spectrophotometer after sonication and use 50 micrograms per IP, though I've had it working with as low as 5 micrograms.

It does rather depend on your antibody and setup as to how much to use as input. There will be a range of inputs you can use with your particular antibody for which the recovered immunoprecipitation signal correlates with the amount of input chromatin.

Assessment of the chromatin concentration after sonication by spectrophotometer is easy. Use the dsDNA setting, and turn the 320nm turbidity correction ON. Yes there is some interference from proteins cross-linked to DNA so the values you get are likely to be slightly out, but I've still found this a far more accurate determination for reaction inputs than cell counting in a side by side comparison.