ANSWER 1: Using too much antibiotic for single-copy markers is the most common reason for not seeing any knock-out recombinants. These are the levels we find typically work well. We have found, however, that using the spectinomycin* cassette to knock out genes can be tricky. The concentration of spec needed to allow selection and prevent background growth must be determined for each construct in each strain.
| ANTIBIOTIC (µg/ml) | SINGLE COPY | MULTI-COPY PLASMIDS |
| Ampicillin | 30 | 100 |
| Kanamycin | 30 | 50 |
| Chloramphenicol | 10 | 20 |
| Tetracycline | 12.5 | 25 |
| Spectinomycin | 30-100* | 100 |
| Hygromycin | 200 | 200 # |
| Blasticidin | not determined | 50 |
# We originally used 50µg/ml hygromycin but found that it gave a high background. We now use 200µg/ml. Hygromycin should be filter-sterilized before addition as the manufacturer does not guarantee sterility.