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Dear all,
I am performing ChIPseq on overexpressed TFs in Drosophila wing discs. After x-linking (1% FA, 10 min) I have the problem that a two step lysis does not work anymore. Basically I am only disrupting the wing discs and lysing the cells during sonication. One of my presumed problems is now, that there might be a lot of unbound (cytosolic) TF, which I precipitate and I want to decrease this precipitation of "useless" TF. Therefore I thought to precipitate/isolate the whole chromatin before I do the ChIP. Does anyone know a good strategy how to do this? Would using spermidine do the job?
Thanks for your help,
Claudia
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Phenol:Chloroform:IAA vs Chloroform:IAA
07 December 2012 - 03:01 AM
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
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
