gandhi

Hi all,

Thank you for the information! I haven't noticed a difference in colony morphology; thank's for the paper.  The galK marker sounds like a great system, but I am already invested in using sacB and have some useful updates.

A colony PCR using CDS primers (amplify gene from start to stop codon) revealed that my isolates have two sized amplicons; once of which is the WT sized band.  To determine if these isolates resulted from a single cross-over (they contain plasmid backbone with a KO copy and WT copy) or a double cross-over (they contain a clean KO but there is a second WT copy of the gene) I amplified sacB.  The isolates do not contain a sacB gene as per colony PCR.  This indicates the presence of a second copy of the gene, which I know was probable from the literature.

I am thinking to repeat the process once I remove the Gentamycin marker from the KO copy.  I will remake a KO cassette that targets the internal region of the gene (that is now only homologous to the remaining WT copy since this region has been deleted fom the KO copy).  Does anyone else have any ideas?

Thank's again.

Hi,

If I am understanding you correctly, your gel shows a WT band and a smaller knock-out band (DCO).  To me, it would seem that your plasmid is still integrated and the isolates are still merodiploid.  How did you know they were merodiploids in the first place?  Also, how did you do your sacB selection?  Did you plate?  Grow culture, then plate?  What sucrose% did you use?