2 replies to this topic

[crockel]

Hey guys,

I am having a pretty tough time to purify DNA after ChIP. So far phenol:chloroform:isoamylalcohol was doing ok, but somehow not anymore. Furthermore I have been asked to omit phenol because it would interfere with the library preparation prior to sequencing.
However, now I was wondering whether I could also do a chloroform:iaa purification of the DNA as well? What is the benefit of additional phenol? The "newer" generation of biologists (with which I mean post-docs and PhD students in our lab) do not have any idea
Thanks a lot for your help!


PS: I have tried many many kits to purify DNA (silica based, and magnetic beads) but they just do not do the job as good as PCIAA does! The loss is just too high. So this is not any alternative

[phage434]

Phenol denatures proteins.  Chloroform dissolves lipids.  I think you are on the right track with using PCIAA in your prep.  I would strongly recommend that you do a chloroform only extraction after the phenol, which will remove almost all of the phenol from your supernatent.  A precipitation and wash and you're set.

[crockel]

thanks a lot phage!