Protocol Online logo
Top : Forum Archives: : Molecular Biology

Hybridization Buffer Won't DISSOLVE - (Dec/07/2007 )

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.

How do I overcome this?

I also noted that the hybridization buffer is basically a modified SSPE with Denhardt's and SDS. A conventional 20x SSPE is 3 M NaCl, 20 mM sodium phosphate, 20 mM EDTA, so I am not sure if the paper had a printing error in the phosphate concentration (although I checked with the original reference and it is correct).


Double check that your phosphate buffer is sodium rather than potassium phosphate. Potassium dodecyl sulfate precipitates out, so adding SDS to a potassium containing soluiton will precipitate.