How to obtain pure DNA from ChIP - (Oct/25/2007 )

Hi All,

I appreciate your input. I found I have never ever able to get a good quality of chip DNA after PCI/CI/EtOH precipitation. The OD260/280 ratio is always about 1, instead of 1.8. However the input DNA that didn't go through antibody immunoprecipitation is always good quality, OD260/280 equals to 1.8. Can someone explain why? and how to improve the purity of chip DNA. Thanks.

hk

-hkwong-

Just a guess: There is residual antibody in your precipitated DNA. Try to clean it up on a column or phenol-chloroform. Of course if the amount is really low, it is not an option.

-Kupac-

I think the problem may lies in reverse crosslinking. How long is your reverse crosslinking? 16 hrs is the minimum, it can be longer. Have you done a proteinase digestion?

Alternatively, you can use Qiagen PCR purification kit to purify your DNA after the proteinase and RNAse digestion. It saves you time and trouble.

-pcrman-

pcrman: Thanks a lot. It is first time to know that reverse time needs 16 hours? Do you think still need proteinase before using Qiagen PCR purification kit to purify your DNA?

-may_2007-