'Tandem' Primers for PCR Mutagenesis

Oliver Sorgenfrei oliver at molgen.biologie.uni-marburg.de
Mon Apr 11 06:17:50 EST 1994


>Hi! If you do two seperate  PCR each with one universal and one mutagenic 
>primer and manage to get the  two bands  of interest. Then purify the bands 
>from  the agarose gel with Qiagen or Geneclean, add DNA from each recovered 
>band to Another set of PCR with the two universal primers. If the condition 
>of PCR is adjusted o.k. You won't have problem to get the mutated product. 
>Good Luck!  Langtuo Deng


Hi,
I made just the procedure mentioned above - worked fine. But upon 
construction of the internal primers be aware that Taq adds an additional A  
to the 3' end.
By the way I would recommend to use one of the thermostable polymerases with 
proof reading function. Otherwise you might get more than just the desired 
mutations. An other advantage ist, that these enzymes do not add an extra A. 
The disadvantage is, that it took in my hands quite some time to find the 
correct parameters for the PCR. 
Good luck
Oliver

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