FAQ 1: How much antibiotic should I use for chromosomal/BAC (single-copy) or plasmid (multi-copy) constructs?

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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. 

 © RECOMBINEERING 2016