Ethanol Precipitation ChIP DNA troubleshoot - (Jul/26/2012 )
I was trying to purify some ChIP DNA following several histone modification IPs. After reverse crosslinking the DNA fragments, I added RNaseA to it, incubated for at least 30 minutes, added Proteinase K with Tris and EDTA and incubated at 45C for over 2 hours. This procedure/sequence of steps has worked before and it's the protocol I follow. Following this, I did a phenol:chloroform:Isoamyl alcohol extraction, followed by a chlorofom extraction. Then I proceeded to precipitate the DNA using glycogen (2µL at 5µg/µL) and 2.5 volume of 100% EtOH. I saw goop precipitating in some of the tubes I was working with. I treated all the tubes exactly the same, so I cannot comprehend why some of the tubes formed this goo precipitate where as the rest did not. After centrifuging, I removed the EtOH supernatant from the tubes and let the pellet dissolve in 0.1X TE. I'm thinking about repeating the RNase A, Proteinase K treatment and the Phenol chlorofom extraction. My question is, when repeating the process again, should I be adding more glycogen for the DNA to precipitate, more salt, etc.?! How else do you think I can recover my DNA fragments? Will repeating any of the process I listed above affect my yield?!
Thank you so much for your input!
You can use PCR cleanup kit to purify your DNA which is much easier and can give you more consistent results. Take a look at this thread http://www.protocol-online.org/forums/topic/11858-purify-chip-dna-using-qiagen-spin-filter/