PCR + KO TARGETING VECTOR

Frauke Naumann fnaumann at scan.genetik.uni-koeln.de
Wed Feb 2 07:53:32 EST 2000


VINCENT A. FUNARI schrieb in Nachricht <388C92E9.E944E3E1 at bio.bu.edu>...
>Does anyone know how PCR errors in a targeting vector would effect
>recombination frequency.
>
>I have an SV129 mouse Genomic phage clone, that I have been unable to
>subclone.
>
>I have experience "PCRing" the targeting region and would like to PCR
>and subclone this for the targeting vector.
>With pfu or some of the other higher fidelity Taqs, I could achieve an
>error rate of less than 1/500 bps.
>
>There might be 20 nucleotide differences in a 10 kb insert.
>
>Any references or experience with using PCR to generate KO targeting
>vectors would be greatly appreciated.
>
>Thank you in advance for your time,
>Vincent Funari
>
>Ph.D. Candidate in MCBB
>Boston University
>353-5311, vfunari at bio.bu.edu
>


Hi, depending on the region of homologous recombination it can matter a lot,
but it must not (like using non isogenic DNA for cloning). Usually, when
using PCR for the construction of the targeting vector, one should sequence
the vector and try to avoid errors in functionally important regions - if
known. If not you would not be sure whether the phenotype observed will stem
from the targeting or from errors in the targeting vector. Why can't you
subclone the clone you have?

Hope that helps
Frauke






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