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Ethanol Precipitation ChIP DNA troubleshoot

DNA EtOH Precipitation Proteinase K RNase A Glycogen

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#1 lobbytruckers

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Posted 26 July 2012 - 03:26 PM

Hello.
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! :)

#2 pcrman

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Posted 27 July 2012 - 04:20 PM

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-...en-spin-filter/





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