PCRs no longer working. HELP!

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Wed Mar 22 04:14:16 EST 1995


In article <Pine.DYN.3.91.950321212910.2531A-100000 at uxa.cso.uiuc.edu>, hodges bradley l <segdoh at uxa.cso.uiuc.edu> writes:
>Maybe its your PCR machine.
>
>On Thu, 16 Mar 1995, John Compton wrote:
>
>> I have been working for 1 1/2 years on a project involving mapping and,
>> now, sequencing of blood and buccal DNA samples from patients.  Initially,
>> I experienced no problems amplifying these DNAs for microsatellite
>> analysis and for template synthesis prior to sequencing.  Now, however, a
>> dark cloud has appeared and only  specific, primarily buccal, DNAs will
>> amplify. 


You did not mention, if you have been using the same cycler all the time, and
what kind of. Our old cycler, which had no "mock-tube" control, slowly went off
control after 3 or so years of regular use. When we installed a separate 
thermocouple and watched the actual temperatures, they were about 3 degrees off
particularly the denaturation temperature. I know now this may affect the yield
of PCR substantially, partilularly you are not expected to vary the
denaturation temperature... I hope this helps, 
*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
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CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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