Media Amendment Stock Solutions

Reagent Abb Stock Conc. Working Conc. Dilution Solvent MSDS Notes
Ampicillin Amp 100 mg/mL 100 µg/mL 1,000× ddH2O link  
Anhydrotetracycline ATC 0.1 mg/mL 100 ng/mL 1,000× DMSO link Can also make a 1mg/mL stock and dilute 1:10 for a final concentration of 0.1mg/mL
3′-Azido-2′,3′-dideoxythymidine AZT 10 mg/mL 200 µg/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 µg/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 µg/mL 1,000× 50% EtOH link  
Chloramphenicol Cam 20 mg/mL 20 µg/mL 1,000× EtOH link  
Cycloheximide Chx 100 mg/mL 100 µg/mL 1,000× 100% DMSO link Anti-fungal, toxic to humans, use PPE!
D-Cycloserine Dcs 4 mg/mL 4 µg/mL 1,000× ddH2O link Degrades noticeably over the course of 1 week at 4°C
Diaminopimelic acid DAP 60 mM 0.3 mM 200× ddH2O link Use 5 µl stock per 1 ml of media. Dissolve ~30min shaking at 37C
Erythromycin Ery 250 mg/mL 250 µg/mL 1,000x EtOH link May crystallize when added to liquid culture. Still works. Selection strength may vary in non-model bacteria
Gentamicin Gen 20 mg/mL 20 µg/mL 1,000× ddH2O link For non-canonical experiments:
Use 20 µg/mL in culture from 20mg/mL 1000× stock
Isopropyl β-D-1-thiogalactopyranoside IPTG 0.1 M 0.1 mM 1,000× ddH2O link Light sensitive, MW: 238.31 g/mol
Kanamycin Kan 50 mg/mL 50 µg/mL 1,000× ddH2O link Plates start to lose sensitivity after 2 weeks; recommended 4C storage time is ~1 month
Nalidixic Acid Nal 30 mg/mL 30 µg/mL 1,000× 1M NaOH link  
Novobiocin Nov 200 mg/mL 200 µg/mL 1,000× ddH2O link  
Rifampicin Rif 50 mg/mL 100 µg/mL 500× MeOH + drops of 10M NaOH link Toxic to humans, use PPE!
Spectinomycin Spec 60 mg/mL 60 µg/mL 1,000× ddH2O link Common to see background growth of cells on selective plates. Best selection with twice the usual concentration of antibiotics (2×).
Streptomycin Str 100 mg/mL 100 µg/mL 1,000× ddH2O link  
Tetracycline Tet 10 mg/mL 10 µg/mL 1,000× 70% EtOH link Light sensitive, stability < 2 weeks
Triphenyl Tetrazolium Chloride TTC 5% (50 mg/mL) 0.05% (50 µg/mL) 1,000× ddH2O link Used in growth/sugar-fermentation indicator agars. Filter sterilize. Light sensitive. Store stock solution at 4°C wrapped in foil. Use at ten-fold higher concentration in motility agar (0.5%).
Zeocin Zeo 10 mg/mL 10 µg/mL 1,000× ddH2O link Some labs report using a higher concentration for selection.
10 µg/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 µm 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 -20°C 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 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. 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 -20°C and 5 at 4°C, compared to what was found on 1 day old MG-Dcs.
  • Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 weeks.

Non-canonical Amino Acid Stock Solutions

Amino Acid
(Abbreviation)
CAS Number Solvent MW
(g/mol)
[Stock] [Stock]
(mg/mL)
[Culture] Notes
Y 60-18-4 Water 181.19 10mM 1.812 250µM Requires heating to 70ºC to dissolve
Stock precipitates out of solution at or below room temperature
IodoY 70-78-0 Water 307.09 10mM 3.071 250µM Dissolves at room temperature with ~1 hour of shaking
AminoY (FAA) 300-34-5 196.203
AminoY (HS) 23279-22-3 Water 287.14 10mM 250µM Dissolves readily without shaking or heat
BromoY 38739-13-8 Water 260.0846 10mM 250µM
ChloroY 7423-93-0 Water 215.63 10mM 250µM
NitroY 621-44-3 Water 226.2 10mM 2.262 250µM Will dissolve with heating to 50ºC, no shaking
ONBY (FAA) 184591-51-3 316.31 Light sensitive
ONBY (S) 207727-86-4 50% DMSO
50% Water
(exothermic)
352.77 50 mM 250µM Light sensitive
Requires adding OH- to dissolve
AzF 33173-53-4 10% DMSO
90% Water
206.204 10mM 2.062 250µM Light sensitive
Requires heating and shaking to dissolve
CNF 167479-78-9 Water 190.2 10mM 1.902 250µM Dissolves at room temperature with ~15 minutes of shaking
L-DOPA 59-92-7 Water 197.1879 10mM 1.972 250µM Oxidizes quickly and turns black in solution
5HTP 4350-09-8 Water 220.2246 10mM 2.202 250µM

FAA: free amino acid
S: salt
HS: hydrated salt
Topic attachments
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Microsoft Word filedocx Making_stock_antibiotics_and_other_reagents.docx r1 manage 89.7 K 2019-01-11 - 21:56 KateElston Easy spreadsheet that tells you exactly how to make the most common stock chemicals
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Contributors to this topic Edit topic JeffreyBarrick, CraigBarnhart, AurkoDasgupta, SeanLeonard, DanielDeatherage, GabrielSuarez, AlvaroRodriguez, JordanMonk, ZuberiAshraf, KateElston, DaciaLeon, SimonDAlton, ElizabethRobinson, BrianRenda, IsaacGifford, DennisMishler
Topic revision: r54 - 2019-07-16 - 18:36:01 - Main.DanielDeatherage
 
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