Chromatin Immunoprecipitation (ChIP) - advice

Syke

Has anyone done one?

I've a student trying to optimise at the moment and I'll fully admit I'm not great at molecular technique theory (I view it akin to witchcraft, if Harry Potter had attempted site-directed mutagenesis I wouldn't have thought anything of it).

We've nice clean genomic DNA in high yields, the antibodies were good and our primers are good.

The problem, it seems, is that genomic DNA is very sticky and our bead and IgG controls are seeing amplification.

Any ChIP experts here who can give advice?

tenandtracer

Are you blocking the beads? If not try this.

Mooch

Without seeing your protocol, it's difficult to try to trouble shoot. As tenandtracer said, blocking is very important. You could also try increasing the stringency of your washes following your IP, by varying the salt/detergent concentration.
Alternatively, you could try to use dot blotting and southern hybridisation as a read-out instead of PCR.

Syke

Mooch wrote: »

Without seeing your protocol, it's difficult to try to trouble shoot. As tenandtracer said, blocking is very important. You could also try increasing the stringency of your washes following your IP, by varying the salt/detergent concentration.
Alternatively, you could try to use dot blotting and southern hybridisation as a read-out instead of PCR.

Thanks for this.

The blocking was fairly stringent. It was actually an issue of gDNA vs cycle number (and primerdesign to some extent). Getting nice readouts now.