Can I make the bacteria competent, then freeze them for later use? According to others, freezing the bacteria results in ~10-fold drop in recombination efficiency, thus we usually use fresh bacteria. For small inserts, that may be acceptable. If you want to try it then instead of washing with water, wash with 10% glycerol then freeze after the washing steps.
What other selection systems are compatible with the AdZ system? We supply the AdZ vectors in SW102 bacteria, so in addition to the SacB/LacZ/Amp system that we use, you can also use GalK selection. We've also successfully used genebridges RpsL/neo cassette.
What can cause the recombineering to completely fail? We’ve had reports from collaborators that the following can cause recombineering to fail:
Growing the bacteria containing the BACs in high salt (10g/L) LB instead of low salt (5g/L) LB. (Note: this applies to liquid LB, not the sucrose agar plates, which are made with no salt atall).
Growing the bacteria in high concentrations of Amp (100ug/ml instead of 50ug/ml).