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FAQ 8: I get tons of recombinants but when I analyze them, none are in the right place.  What is wrong? 

Answer 8: Did you use a plasmid for the template for your drug cassette? If so, more than likely all of your “recombinants” are just transformants that contain the template plasmid. Electroporation is a very efficient transformation method and supercoiled plasmid DNA transforms very well. We recommend not using plasmids as templates for this reason and we use chromosomal templates whenever possible. We have a stain called, “T-SACK”, that is available that can serve as a template for tetA, kan, tet-sacB, amp and cat cassettes. We highly recommend using it as your template. The best source for amplification of the cat-sacB seletion/counter-selection cassette is the bacterial strain TUC01, which is available from the Court laboratory. Although the plasmids pKO4 and pELO4 contain cat-sacB, because they are plasmids, they are problematic as templates.