dextran sulfate-- a cheaper source??
afc at gnv.ifas.ufl.edu
Tue Apr 12 11:34:28 EST 1994
In article <1994Apr12.124209.1 at hkucc.hku.hk>, h9290184 at hkucc.hku.hk writes:
> In article <Cnt28w.IuG at news.cis.umn.edu>, mman at molbio.cbs.umn.edu (Michael Man) writes:
>> I use dextran sulfate in my hybridization solution. It gives me very
>> good results, but it is too expensive, $732 per 500 gms from Sigma. Does
>> anyone know a cheaper place to go? Thanks for your help.
>> Also I will be very grateful if you can suggest an alternative way
>> to prepare prehyb. and hyb. solution. I use GeneScreen transfer membrane and
>> UV-crosslink RNA.
>> mman at molbio.cbs.umn.edu
> Hi there,
> Sometimes ago in 1989 at UCSF, I mentioned dextran sulfate in my hyb solution
> to someone in the next door lab, he answered me with "Do you have dinosaurs for
> pets at home?" That was before Jurassic Park.
> I now use 7% SDS and .25 M Na2HPO4 pH7.2 for my prehyb and hyb solution and
> nothing more for all my blots (north/south-erns) or colony/plaque screening for
> all nylon membranes only not for nitrocelulose.
> Kaimin Chan
> the Institute of Molecular Biology
> the University of Hong Kong
> Hong Kong
> H9290184 at hkucc.hku.hk
Useful things to put in hybridization solutions:
Dextran sulfate is used to accelerate the hybridization, presumably by
tieing up most of the free water in the hybridization solution. This can
be important when probing for single copy genes in large genomes or rare
messages on northerns. Otherwise leave it out, as it is expensive.
SDS is used to prevent background binding of probe to the filters. It
can be used to substitute for Denhardt's or carrier DNA. It does *not*
substitute for dextran sulfate. Nonfat dry milk can help reduce background
also, but don't use it with northerns!
Formamide can be used to lower the Tm of the hybrid so that the hybridization
can be done at a more convenient temperature.
It is also a good idea to put in some buffer (tris or PB are usually used),
EDTA to chelate any divalent cations that may have made their way
into the hybridization, and high Na+ to speed up the reaction. 6x SSPE from
Maniatis is cheap and effective.
I prehybridize in 2% SDS, 1% nonfat dry milk and hybridize in 6x SSPE,
2% SDS, 2% nonfat dry milk, and formamide if needed.
More information about the Methods