Posted 22 August 2009 - 02:26 PM
Posted 22 August 2009 - 04:52 PM
I am having trouble with my ChIP assay...I do end up with a white DNA pellet at the end but after running PCR with primers that our lab uses (others have already optimized the number of PCR cycles), my agarose gel shows no bands, even for my total chromatin samples. Any ideas why this might be? Thanks!
How do you clean-up the DNA after the IP (spin columns, phenol:chloroform and ethanol precipitation, Fast ChIP, etc.)? How do you prepare the total chromatin samples prior to running PCR (do you follow the same clean-up protocol as your IPs) or if you don't clean them up, do you at least dilute them in a PCR compatible buffer prior running PCR?
Posted 22 August 2009 - 05:46 PM
Posted 23 August 2009 - 05:56 PM
I use chloroform followed by 100% ethanol for both my IP samples as well as my total chromatin samples. I've noticed that after adding chloroform and centrifuging, as I am taking out the supernatant on ice, there seems to be something that is precipitating out and forms a layer at the top of my supernatant. Not sure what this is or if it is protein contamination so after obtaining my DNA, I even use a PCR purification kit before running PCR...
My suggestion is to use a MinElute PCR clean-up kit (or other low retention/low capacity clean-up kit) to clean-up your DNA and skip the phenol:chloroform and ethanol precipitation altogether.