very low ChIP'd DNA amount. (View forum version)


Posted 16 December 2009 - 02:50 PM

Hi, I tried nuclei lysis on my ChIP samples, but I got very little amount (1-2 ng from 2-3 million cells) :lol: . Does it mean a bad ChIP? What is the normal amount of ChIP'd DNA?
And I noticed that SDS in my samples kept being precipitating down during my sonication step. Is it normal? Oh~~~~, ChIP is torturing me!!

Thank you very much for your replies!!


Posted 19 December 2009 - 07:44 AM

:lol: so I guess you mean that you have a total of ~2ng pulled down DNA for your given antibody after ChIP? To be honest, I never really bothered to measure the DNA amount in the IPs (too precious, and unless you're using picogreen or something similar probably too low a concentration to get reliable OD readings). It does sound a little on the low side though...

Do you get amplification of a positive control region over your negative / background control? As for numbers, I usually get around 400ug sonicated DNA from ~5mio nuclei (HT1080) and use ~100ug DNA per IP (only measured these quantities a couple of times, otherwise normally just go with cell number / volumes). From that, I'm usually getting specific PCR amplifications in the order of 1-10% (vs. 0.01-0.1% for unspecific IgG) of the Input depending on the antibody (histone mods).

As for the SDS precipitation: what's your concentration in the sonication buffer? You might just reduce it if it's 1% - for nuclei, 0.2% is plenty already...