This seems to be a very important decision to make, usually, in terms of saving time at least. We have tried hygromycin, zeocin, geneticin and puromycin. Among these, puromycin seems to kill non-resistant cells the fastest, and bacause it is used at a very low concentration, it is also very cheap. Geneticin (G418) seems to be the worst , taking weeks, and a lot of people seem to face problems with it. The concentration used is fairly high, and the worst thing seems to be that most of the manufacturers seem to cell a lot of expression vectors with G418 resistance genes! They reserve puromycin exclusively for lentiviral vectors, etc, the logic behind which I cannot understand.
Zeocin is OK, but takes some time, and its mechanism of killing means that it takes some effort to tell which cells are actually dying off and which are not. Hygromycin seems to be fairly fast and non-ambiguous.
One antibiotic we haven't used is blasticidin. Has anyone used blasticidin for selection? How many days does it take to get colonies of resistant cells, and is it a good, problem-free antibiotic for selection?