[Q] ThermoSequenase aging

Eric Kofoid 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:
http://www.bc.biol.ethz.ch/BiochemistryIII/                                

-> CH-8092 Zurich                                     /Sasha/kraev.html

Alexander --

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.

Cheers,

Eric.

-- 
Eric Kofoid        Dept.Biology, U.Utah       kofoid at biology.utah.edu



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