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problem with gene knockout by homologous recombination: crossover always happen

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#1 Lynn Shay

Lynn Shay


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Posted 22 March 2013 - 06:02 PM

we are trying to knock out some non-essential genes in V. alginolyticus using inframe deletion. basically we cloned the upper stream and downstream 800-bp homology into a suicide vector and this vector is transferred into V. alginolyticus by E. coli SM10lambdapir. when we use PCR to verify the integration of this suicide vector onto the chromosome, we found that all the transconjugants integrated the plasmid at the same homology (we tested a total of 11), while theoretically it should be 50/50. we checked that both homologies are of about the same size, ~800 bp, with the same GC content, 46% or 48%, and the suicide plasmid was sequenced to confirm we have the right homology. not surprisingly, when we select the 2nd crossover, all of them were wild type.Posted Image
so it seems as if the crossover always happens at the same homology...any idea why this happened? is there anything else we should check?
Thanks in advance for any suggestions.

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