FAQ 7: Why don’t I get any recombinants?

Answer 7: There can be several causes but the most common are 1) using the wrong drug concentration for single-copy cassettes and 2) losing too many cells during preparation of recombineering- and electroporation-competent cells. Check your drug concentrations under FAQ 1. If your viability counts are very low (<107), this could indicate that electroporation killed a lot of cells or more likely, you lost a lot of them down the sink. The pellet is very soft after the cells are in H2O and care must be taken when decanting.

    Another common problem with not getting recombinants is not allowing enough outgrowth time to allow 1) expression of the drug-resistance gene and 2) segregation of chromosomes (newly made “wildtype” away from ones still containing cat-sacB) if you are doing a counter-selection such as cat-sacB. In both cases, at least a 2-3 hour outgrowth is necessary. See Protocols.