How to sequester SDS in order to do ligation? - (Apr/07/2006 )
I am trying the chromatin conformation capture (3C) procesure.
In short, I crosslink chromatin and digest cross-linked chromatin with RE. The restriction enzyme is inactivated by addition of 1.6% SDS and incubation at 65 degree. The protocol says that you have to add 1% trtion x-100 in order to sequester SDS and allow the following reactions. The sample is then 40x diluted and ligated. Now, the problem is that ligation doesn't happen. Ligase works fine in normal condition and in the presence of 1% triton x-100, but no in the presence of SDS and 1% trtion x100.
Is 1% triton x100 good for sequestering SDS?
Any tips on doing ligation in the presence of SDS and triton x100?
I can't help you with the SDS + ligation question, but I can tell you that the triton X-100 will make transformation of your ligation mixture into chemically competent cells extremely inefficient. You should either electroporate (haven't tried that) or ethanol precipitate the ligation mixture (this works).
Thanks for the reply. I am not going to do transformation using the ligation. It will be used for PCR.
If you're going to use the DNA for PCR, I would suggest precipitating the SDS by adding an equal volume of 1M potassium acetate pH4. Centrifuge to pellet the SDS, then remove the supernatant to a fresh tube and add 3 volumes of ice cold ethanol to precipitate the DNA. Spin, wash, etc, resuspend in water, PCR, voila
I thought potassium was a better precipitant for SDS than sodium. I'd try potassium acetate (again at pH 4 or pH 5).
nucleospin columns have a procedure for purifying components in SDS containing solution.
You're absolutely right, I meant to put potassium
Obviously trying to swap out sodium for sodium isn't going to be very successful