You mention that the CatR+ host bacteria (without the SacB-containing plasmid) died when it was grown with 10% sucrose and chloramphenicol. This is not a 'good' result from what I understand. To use this protocol, a cell should only die in sucrose if they contain the SacB gene from the plasmid. Why did your host bacteria die with sucrose, if not due to the SacB gene?

Try Gibson cloning. It saves time and money, and is more simple that all of this.

@Nicole Wheatley: I believe the system the poster is using is an antibiotic free selection system where plasmid transformation suppresses expression of chromosomal sacB leading to survival on sucrose. This isn't my field but a lot of regulatory agencies don't like the idea of using antibiotic selection for anything that might be used for human consumption, so there are a lot of systems out there based on conditionally lethal genes or nutritional selection in minimal media.

@Januar Ishak

Honestly if you have colonies my first guess would simply be that your colony PCR isn't working and you should just try miniprepping some colonies and digesting them with a restriction enzyme, I'd try that before troubleshooting the selection itself. Colony PCR is a quick and dirty method and it's pretty failure prone.

@alexandra Johnson: Yes, I now see that he says the SacB is on the host chromosome. I've only ever seen SacB systems with SacB in the plasmid.

Sorry for the confusion Januar Ishak~!