Stability of stored PCR Templates?

tweissen at my-deja.com tweissen at my-deja.com
Thu Oct 5 03:57:29 EST 2000


Common polypropylene and -ethylene tubes can adsorb considerable
amounts of DNA, especially short fragments at relatively high ionic
strenght, i.e. PCR products(C Gaillard, F Strauss. Intern.
Biotechnol. Laboratory 2000 Aug: 6).
In addition, these surfaces catalyze the formation of high molecular
weight aggregates (Biopolymers 1999 Dec;50(7):679-89) which are perhaps
refractory to amplification.
Of course this could only happen in solution, i.e. if your samples had
been thawed many times during the storage period. You could try low
binding propylene tubes (http://www.axygen.com/pages/maxymum.html, have
not tested them and no affiliation!), or tubes made from polyallomer.
It may be possible to recover some of the bound DNA by diluting the
sample, adding Triton X-100 to 0.1% and heating.

Good luck!

Thomas

Thomas Weissensteiner
Edward Jenner Institute for Vaccine Research
Compton RG20 7NN, U.K.
T.:   0044 1653 577900 x3931
Fax: 0044 1635 577901
Email: tweissen at replace.this.with : hgmp.mrc.ac.uk
Edward Jenner Institute for Vaccine Research: http://www.jenner.ac.uk/
PCR Jump Station:  http://www.highveld.com/pcr.html




In article <492C5.29679$ib7.4184312 at news1.rdc1.nj.home.com>,
  "Paul Mitsis" <pmitsis at seqltd.com> wrote:
> Hi All,
>
>     We've been running a series of PCR experiments on  approximatelty
100 bp
> DNAs with potentially a lot of secondary structure. Recently our
stored
> templates have become more and more refractory to re-amplification.
These
> are purified from PCR reactions (Qiagen) and stored at -20oC in TE.
My
> guess was aggregation but none  of the simple things to try to
dissagregate
> them (heat or urea) seems to help.  Has anyone seen this before or
have a
> suggestion what might be going on.
> Thanks for any help you can give.
>
> Paul Mitsis
> Senior Scientist
> Praelux Inc.
>
>


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