[Q] ThermoSequenase aging
kofoid at biology.utah.edu
Tue Jun 25 12:08:37 EST 1996
In article <4qmhib$c2i at elna.ethz.ch>, Alexander Kraev
<kraev at bc.biol.ethz.ch> wrote:
-I have used both kits US78500 (radioactive) and RPN2438 (fluorescent),
-as well as polymerase separately with homemade termination mixes and
-did not notice that it is a particularly unstable enzyme. It is
-definitely more tolerant to clumsiness and general abuse than
-Sequenase V2.0. The problem you describe looks like that of a reduced
-polymerase activity, but this reduction may come not only from "ageing",
-but also from other, i.e. physical factors, such as an erratic cycler.
-We had two years back a problem with regular PCR, when everybody started
-getting smears instead of bands and it was traced to the cycler being
-offset by 3-5 degrees at denaturation temperature. Did you get the same
-problem with the control DNA included?
-Alexander Kraev, PhD Internet:
-kraev at bc.biol.ethz.ch
-> Lab.of Biochemistry III Phone: 0041-1-632-31-47
-> Swiss Federal Inst. Of Technology FAX: 0041-1-632-12-13
-> Universitaetsstr. 16 Home Page:
-> CH-8092 Zurich /Sasha/kraev.html
Good point. I have been assuming that the cycler is in good adjustment
because it does good PCR. However, I'll take a closer look at its
calibration and definitely check out the control primer/template pair.
Thanks for the advice.
Eric Kofoid Dept.Biology, U.Utah kofoid at biology.utah.edu
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