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BAC Recombineering PCRscreening

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#1 jimmykjm



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Posted 07 July 2011 - 07:18 PM

Hello. I am currently doing BAC recombineering in which a kanamycin resistance gene cassette flanked by homology arms is inserted via homologous recombination into pBeloBAC11. I use electroporation to transform DH10B carrying pBeloBAC11 and pGETrec (helper plasmid that expresses the recombineering genes recT, recE and Gam, under an arabinose inducible promoter). I get plenty of colonies that are kanamycin and chloramphenicol resistant.

However, during the final PCR screening in which I use primers that binds to regions flanking the insert site on pBeloBAC11, I always get mixed results, in which the long fragment (signifying recombinant) and the short fragment (non-recombinant as gene not inserted) are present! Most of the time, the short fragments are brighter and stronger than the long one. Only about 1 in 10 will be true recombinants (only the long fragment present).

I feel that it is unlikely to be template contamination as I gel purified the cassette and DpnI digest it two times. I also check the sequence of all the plasmids and the genomic DNA to make sure there are no other homologous regions.

So I am kinda desperate to know what went wrong?

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