Second PCR Round Phenomenon

Mark Schweder mschweder at gnv.ifas.ufl.edu
Sun Nov 29 16:36:46 EST 1992


 I have run into this problem on a number of occasions and occurs as 
 follows. I amplify a fragment of DNA by PCR from genomic DNA and then take 
 a small aliquot of the first reaction and add it as the template for a 
 second PCR round. The result of the second reaction is a smear of 
 generally large pieces. The target fragment is no where to be found. I 
 have attempted a number of things to get the reaction optimized, ie, 
 annealing temperature, [dNTP], [MgCl2], [primer], [template], etc. I'm 
 aware of nested primers but haven't tried that. I was just wondering why 
 this second reaction doesn't work with some templates and does with some 
 others? I suspect that the template undergoes some kind of self priming 
 and concatenates itself into long fragments. Then of course the question 
 is why doesn't that occur during the initial PCR reaction?
-- 
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   Mark Schweder                | There are three things that smell like fish!
   Plant Science Laboratory     | One of them is fish...
   Agronomy Department          | The other two...
   University of Florida        | Are growing on you...
   Gainesville, Florida 32611   | (Frank Zappa, Jumbo Go Away)
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           904-392-1890         |         mschweder at gnv.ifas.ufl.edu
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                  DISCLAIMER: I know nothing about nothing.



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