AP-PCR QUESTION

Jenny Williams Jenova at microbes.demon.co.uk
Fri Jul 4 15:10:52 EST 1997


In article <tyou.64.33BB671B at postbox.acs.ohio-state.edu>, "Taek H. You"
<tyou at postbox.acs.ohio-state.edu> writes
>
>>I have recently completed a project on molecular typing using Arbitrary-
>>Primer PCR (AP-PCR). This technique uses a single arbitrary primer in
>>each PCR assay. I used random primers based on published sequences which
>>had previously been applied successfully in the typing of Clostridium
>>difficile. I followed the published protocols carefully, but was unable
>>to get reproducible results.  I then combined the same two random
>>primers that I had previously used in single primer PCR assays, in the
>>same reaction tube. The assay conditions were identical to that of the
>>single primer assays, with the exception that 2 primers were now taking
>>part in the reaction. Also, the total amount of primer in the reaction
>>tube was doubled. This combination produced reproducible results. Also,
>>the resulting DNA profile produced after gel electrophoresis was
>>different from the sum of the DNA profiles of the single primer assays.
>>Can anyone explain these phenomena?
>
>Isn't there any different machine involved?
>When I ran the same reactions in different machines, results were remarkably 
>different.
>In addition, many published stuffs were not reproducible until some twist in 
>PCR.
> I am talking about a regular PCR, not AP-PCR. But, I think that might be 
>the cause.

Hello Taek
Sorry, I obviously need to clarify my post. When I stated that I didn't
get reproducible results, I was talking about not getting the exact same
DNA profile when testing the same strain several times in the same
thermocycler. i.e. my result was not reproducible. I wasn't trying to
duplicate the exact same profiles as that in the published papers, since
my collection of Clostridium difficile strains is different from those
used in  previous studies.
Thankyou for taking the time to reply.
Regards

-- 
Jenny Williams



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