dNTP problem (and magnesium)->gradients

Bradley Turner bsturner at mbcrr.harvard.edu
Tue Sep 5 10:52:29 EST 2000

Hello Duncan,

Do you have a reference for the "freeze-thaw" sucrose gradient
method?  I don't think that I've come across that before, but
it sure sounds interesting!  Does it work for cesium gradients 

Brad Turner

                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 605                   bsturner at biosun.harvard.edu
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Boston, MA 02215                bsturner at mbcrr.harvard.edu

On Tue, 5 Sep 2000, Dr. Duncan Clark wrote:

> In article <OF953379A1.5E172E74-ON87256950.00798779 at nexstar.com>,
> tfitzwater at gilead.com writes
> >While   many   sources   indicate   that  magnesium  stock  solutions  form
> >concentration  gradients  during  multiple freeze/thaws,
> That is also a great way of producing sucrose gradients. Start with
> 12.5% and do three slow (in a fridge) freeze thaws and you will generate
> a nice low to high gradient for centrifuging in swing out rotors etc.
> > C. Y. Hu, M. Allen
> >and   U.  Gyllensten  1992  PCR  Meth.  Appl.  2:182  reported  performance
> >variability  of  PCR  reaction buffer solutions containing magnesium.  Free
> >magnesium  changes  of  0.6  mM  observed  by  them  dramatically  affected
> >amplification  efficiency in a allelic specific manner.  Heating the buffer
> >at 90°C for 10 min restored the homogeneity of the buffer, supporting their
> >hypothesis  that  magnesium  chloride  precipitates as a result of multiple
> >freeze/thaw cycles.
> I'll go and have a look at that. I suppose with users using chemical
> Hotstart, the 10mins activation at 95C would mean one would never see
> this variability.
> Many thanks
> Duncan
> -- 
> The problem with being on the cutting edge is that you occasionally get 
> sliced from time to time....
> Duncan Clark
> DNAmp Ltd.
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