Media Amendment Stock Solutions

Reagent Abb Stock Conc. Working Conc. Dilution Solvent MSDS Notes
Ampicillin Amp 100 mg/mL 100 g/mL 1,000× ddH2O link
3′-Azido-2′,3′-dideoxythymidine AZT 10 mg/mL 200 g/mL 50× ddH2O link Can substitute pharma grade Zidovudine. Soluble up to 20 mg/mL in ddH2O
Bromo-chloro-indolyl-galactopyranoside X-gal 40 mg/mL 80 g/mL 500× Dimethylformamide (DMF) link X-gal is a histochemical substrate for β-galactosidase,
which cleaves X-Gal to yield an insoluble blue precipitate.
Carbenicillin Crb 100 mg/mL 100 g/mL 1,000× 50% EtOH link  
Chloramphenicol Cam 20 mg/mL 20 g/mL 1,000× EtOH link  
Cycloheximide Chx 100 mg/mL 100 g/mL 1,000× 100% DMSO link Anti-fungal, toxic to humans, use PPE!
D-Cycloserine Dcs 4 mg/mL 4 g/mL 1,000× ddH2O link Degrades noticeably over the course of 1 week at 4C
Diaminopimelic acid DAP 100 mM 0.3 M 333× ddH2O link Use 3 l stock per 1 ml of media. Dissolve with overnight shaking.
Gentamicin Gen 5 mg/mL 5 g/mL 1,000× ddH2O link For non-canonical experiments:
Use 20 g/mL in culture from 20mg/mL 1000× stock
Isopropyl β-D-1-thiogalactopyranoside IPTG 0.1M 0.1mM 1,000× ddH2O link Light sensitive, MW: 238.31 g/mol
Kanamycin Kan 50 mg/mL 50 g/mL 1,000× ddH2O link Plates lose sensitivity after 2 weeks
Nalidixic Acid Nal 30 mg/mL 30 g/mL 1,000× 1M NaOH link  
Novobiocin Nov 200 mg/mL 200 g/mL 1,000× ddH2O link  
Rifampicin Rif 50 mg/mL 100 g/mL 500× MeOH + drops of 10M NaOH link Toxic to humans, use PPE!
Spectinomycin Spec 60 mg/mL 60 g/mL 1,000× ddH2O link  
Streptomycin Str 100 mg/mL 100 g/mL 1,000× ddH2O link  
Tetracycline Tet 10 mg/mL 10 g/mL 1,000× 70% EtOH link Light sensitive, stability < 2 weeks
Zeocin Zeo 10 mg/mL 10 g/mL 1,000× ddH2O link Some labs report using a higher concentration for selection.
10 g/mL is specifically used for growth of amberless strain A37
Working concentrations are for liquid or solid media.

Preparation

Generally, prepare 30-50 mL of solution in a 50 mL conical tube. Then, filter sterilize solutions by pushing them through a 50 mL syringe fitted with a 0.22 m filter made of a material appropriate for the solvent. Aliquot into 1.5 mL portions in 1.7 mL tubes using the repeat pipettor. Label with a permanent marker (one that won't wash off with ethanol). Store at -20C in the dark.

Other considerations

  • Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months.
  • Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4C for about 2 weeks. Stock solutions can be stored at 4C for 2 weeks but can last as long as 4-6 months when stored at -20C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable.
  • D-cycloserine is best used within two days of pouring. A single small-scale experiment found 6-10x as many E. coli colonies on 8-day old MG-Dcs plates that had spent 3 of those days at -20C and 5 at 4C, compared to what was found on 1 day old MG-Dcs.
  • Kanamycin plates only last a few weeks at 4C; best to make fresh Kan plates every 2 weeks.
Topic attachments
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elsedocx Making_stock_antibiotics_and_other_reagents.docx manage 89.7 K 16 Apr 2014 - 18:28 Main.CraigBarnhart How to make stock antibiotic solutions
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Topic revision: r39 - 19 Dec 2016 - 03:54:50 - Main.JeffreyBarrick
 
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