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 
also?

Thanks,
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
330 Brookline Avenue            bturner at caregroup.harvard.edu
Boston, MA 02215                bsturner at mbcrr.harvard.edu
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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.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
> http://www.dnamp.com
> http://www.genesys.demon.co.uk
> 
> 
> 


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