dNTP problem (and magnesium)

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Tue Sep 5 03:19:36 EST 2000


> From:          tfitzwater at gilead.com
> Subject:       Re: dNTP problem (and magnesium)
> Date:          4 Sep 2000 23:15:16 +0100
> Organization:  BIOSCI/MRC Human Genome Mapping Project Resource Centre
> X-To:          methods at hgmp.mrc.ac.uk
> To:            methods at hgmp.mrc.ac.uk

>
> >From: Dr. Duncan Clark (Duncan at nospam.demon.co.uk)
> >Date: Tue 29 Aug 2000 - 09:32:03 BST
>
> >In article <mantei-B47484.08313729082000 at news.ethz.ch>, Ned Mantei
> <mantei at cell.biol.ethz.ch> writes
> >In article <39AB188F.608D3A57 at med.monash.edu.au>, Paul Cullen
> ><Paul.Cullen at med.monash.edu.au> wrote:
> >>Make sure you are not defrosting them in a water bath. dNTPs are
> >>quite sensitive to heat and should be stored at -20 C. Defrost on
> >>ice.
>
> >Although I generally agree that keeping reagents cold is a good
> >idea, dNTPs can't be as sensitive as all that. In the course of a
> >PCR they survive maybe an hour or more at 72 C and half an hour to
> >an hour at 94 C. They can also be shipped at RT.
>
> >>Incidentally, I have heard the claim that MgCl2 doesn't redissolve
> >>easily and needs to be warmed after thawing.
>
> >Never come across that one in many years of producing PCR reagents.
>
> >Duncan
>
> While   many   sources   indicate   that  magnesium  stock
> solutions  form concentration  gradients  during  multiple
> freeze/thaws, 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.
>
> Tim Fitzwater
> Principal Research Associate
> Gilead Sciences
>
>
> ---
>
>
Tim, it's probably not the magnesium chloride itself is
precipitating. It rather forms complexes with other compounds found
in the buffer. The result would be rhat either the effective
magnesium concentration is lower or another compund is not available
in the needed concentration.

Wolfgang

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Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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