Homebrew Hybridization buffers - (Dec/08/2007 )
I am trying to make a certain hybridization buffer. The recipe: 1.5 M NaCl, 40 mM sodium phosphate buffer pH 7.2, 10 mM EDTA pH 8, 10x Denhardt's soln, 0.2% SDS. It's taken from the protocol "Direct genomic isolation" published by Bashiardes et al, Nature methods, 2:63, which uses biotinylated BAC probes to capture genomic fragments of interest.
My problem is that the hybridization buffer does not dissolve. I put in the NaCl, phosphate buffer, EDTA, then SDS stock solutions, and the solution precipitates when SDS is added. The hybridization buffer is basically a modified 10x SSPE with Denhardt's and SDS. A conventional 20x SSPE is 3 M NaCl, 20 mM sodium phosphate, 20 mM EDTA. Same problem happens when I added SDS to the 10x SSPE.
How do I overcome this?
first make a 10% or 20% SDS solution in H2O. Then add it gently (bit by bit) and heat the solution to 37°C should do the trick.
are you using potassium phosphate buffer?
potassium dodecyl sulfate is insoluble. it will form when you add sds to kphos and precipitate (white).
use sodium phosphate buffer, instead.