Paulsson:Antibiotics

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Bacteriostatic vs. Bacteriocidal

MOA, references

always run through a 0.2um filter(to sterilize and remove undissolved antibiotic - especially for single cell work!!!), unless dissolved in a solvent that will destroy the filter

working concentrations depend on copy number


Ampicillin (sodium salt)[1]
stock solution: 100 mg/ml in water (unstable, do not store too long or freeze/thaw aliquots more that 3 times)

Carbenicillin (sodium salt)
stock solution: 50 mg/ml in water (more stable than ampicillin, and six times the price); use at same working concentration as ampicillin

Cephalexin
stock solution: 20 mg/ml in ~60mM NH4OH. Suspend 20g cephalexin in 900 ul water, then add ~60 ul 1M NH4OH. Note: concentrated NH4OH is difficult to pipet accurately. And don't dissolve cephalexin in 1M NH4OH as recommended by Sigma: unstable even when stored at -20deg. MIC is 10 ug/ml.

Chloramphenicol [2]
stock solution: 100 mg/ml in 95% EtOH (don't filter, EtOH will dissolve filter membrane)

  • NB: there are multiple CAT genes in use for conferring resistance to chloramphenicol, and some are much less effective than others. The Tn9-derived CAT gene from pACYC184 supports growth on ~15 ug/ml chloramphenicol, even when integrated on the chromosome. A reduced version (~540bp) of this Tn9-derived CAT gene can be found in other vectors (eg. pJPC12 and pSU series (Gene, v.102, p75-78, 1991)) and supports growth at ~5ug/ml chloramphenicol at low copy number.

For complete inhibition of protein synthesis, as when measuring protein degradation, use 100 ug/ml.

Gentamycin

Kanamycin [3]
stock solution: 35 mg/ml in water
Wikipedia entry for Kanamycin
resistance

Nalidixic acid
stock solution: 50 mg/ml in 50mM NaOH

Neomycin

Novobiocin
stock solution: 100 mg/ml in water (insoluble at lower PH, comes out of solution when added to LB)

Rifampicin
stock solution: 20 mg/ml in DMSO, store at -20°C (or MeOH, store at 4°C, good for 2 weeks)
working concentration: 200 ug/ml
light sensitive

Spectinomycin [4]
stock solution: 100 mg/ml in water
working concentration for selection: 50 ug/ml
single chromosomal copy of resistance gene confers resistance at 40 ug/ml spectinomycin (applied as an overlay), perhaps higher, I didn't test (PerM.) working concentraion for arresting protein synthesis: 150-500 ug/ml

Tetracycline hydrochloride[5]
stock solution: 10 mg/ml in water[6]
working concentration: 10 ug/ml for most strains; some strains (e.g. PMB150) grow very slowly at this concentration, but normally at 5 ug/ml
for chromosomally integrated resistance genes (eg. BL21-AI, PMB474) I generally use 2 ug/ml, but this can be insufficient for selection of transformants
NB: anhydrous tetracycline used for induction of tet promoter - 0.2 ug/ml aTc (aka AHT)

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