Paulsson:Antibiotics: Difference between revisions
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'''Chloramphenicol''' [http://openwetware.org/wiki/Chloramphenicol]<br> | '''Chloramphenicol''' [http://openwetware.org/wiki/Chloramphenicol]<br> | ||
stock solution: 100 mg/ml in 95% EtOH (don't filter, EtOH will dissolve filter membrane) | stock solution: 100 mg/ml in 95% EtOH (don't filter, EtOH will dissolve filter membrane) | ||
For low copy plasmids (e.g. | * NB: there are multiple CAT genes in use for conferring resistance to chloramphenicol, and some are much less effective than others. For low copy plasmids (e.g. R1 and F origins) bearing the CAT gene from pACYC vectors, use ~15 ug/ml chloramphenicol. The same plasmid bearing the CAT gene from pJPC12 should be selected for on ~5ug/ml plates.<Br> | ||
For complete inhibition of protein synthesis, as when measuring protein degradation, use 100 ug/ml. | For complete inhibition of protein synthesis, as when measuring protein degradation, use 100 ug/ml. | ||
Revision as of 08:43, 28 February 2011
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 approx. 60mM NH4OH (concentrated NH4OH is difficult to pipet accurately)(don't dissolve 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. For low copy plasmids (e.g. R1 and F origins) bearing the CAT gene from pACYC vectors, use ~15 ug/ml chloramphenicol. The same plasmid bearing the CAT gene from pJPC12 should be selected for on ~5ug/ml plates.
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)
