Explanation for freeze-thaw cycles that generate sucrose gradient

Thanit Pewnim thanit at su.ac.th
Thu Sep 7 00:13:48 EST 2000


Hello Dr. Clark,

We will greatly appeciate if you would give us a physical basis behind the
freeze-thaw method that generates a sucrose gradient.

Thank you



%----------------------------------------------------------------------%
=09Thanit Pewnim, Department of Chemistry, Silpakorn University
=09Nakornpathom 73000, THAILAND >>>>> Phone +66 34 255797
=09Fax +66 34 271356 or 255820, E-mail <thanit at su.ac.th>
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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  f=
orm
> >concentration  gradients  during  multiple freeze/thaws,
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> 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.
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> > C. Y. Hu, M. Allen
> >and   U.  Gyllensten  1992  PCR  Meth.  Appl.  2:182  reported  performa=
nce
> >variability  of  PCR  reaction buffer solutions containing magnesium.  F=
ree
> >magnesium  changes  of  0.6  mM  observed  by  them  dramatically  affec=
ted
> >amplification  efficiency in a allelic specific manner.  Heating the buf=
fer
> >at 90=B0C for 10 min restored the homogeneity of the buffer, supporting =
their
> >hypothesis  that  magnesium  chloride  precipitates as a result of multi=
ple
> >freeze/thaw cycles.
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> 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.
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> Many thanks
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> Duncan
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> =20
> --=20
> The problem with being on the cutting edge is that you occasionally get=
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> sliced from time to time....
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> 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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