Emergency~~~Storage sonicated ChIP lysate at -80 without centifugation? - ChIP (May/20/2011 )
I wnat to know if anyone keep the sonicated ChIP lysate at -80 degree directly without centifugation. I checked many protocols, they only said "centifugation after sonication and storage at -80." Could it possible to centrifuge the sonicated lysate after confirming the size of the sonicated fragments?
Thanks for answering.
If you don't centrifuge before checking the size of your fragments you will often times get a high molecular weight smear that represents insoluble chromatin that would have been otherwise cleared by the centrifugation step. As long as you can distinguish your "sheared" material from the insoluble, high MW chromatin fraction you can accurately determine the sizes. One thing that you will probably want to do is, after you retrieve your chromatin from the -80 for use, let it warm up to room-temp and vortex lightly to diffuse the salts......otherwise you may precipitate out the fair majority of your chromatin. I think it's safe to say the majority of us clear our chromatin by centrifugation before we freeze it back........but I think it doesn't matter when you do it, as long as you do some sort of clearing step before your IP.
I agree that you need to centrifuge BEFORE you run the gel or Bioanalyzer to check your fragment sizes. The centrifugation step, apart from what chabraha mentioned, is also used to get rid of cell debris, which could interfere with your PCR reaction, as well as with the IP step for the actual ChIP. Somewhat related matter here... You usually end up getting a layer of lipids on top of your sonicated lysate after centrifuging. I wonder if anyone has data regarding whether it interferes with IP in ChIP.