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  
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 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 mM 333 ddH2O link Use 3 l stock per 1 ml of media. Dissolve with overnight shaking.
Erythromycin Ery 250 mg/mL 250 g/mL E. coli
10 g/mL Lactobaccillus sp.
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 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 0.1 M 0.1 mM 1,000 ddH2O link Light sensitive, MW: 238.31 g/mol
Kanamycin Kan 50 mg/mL 50 g/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 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 Common to see background growth of cells on selective plates
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.

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 250M Requires heating to 70C to dissolve
Stock precipitates out of solution at or below room temperature
IodoY 70-78-0 Water 307.09 10mM 3.071 250M 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 250M Dissolves readily without shaking or heat
BromoY 38739-13-8 Water 260.0846 10mM 250M
ChloroY 7423-93-0 Water 215.63 10mM 250M
NitroY 621-44-3 Water 226.2 10mM 2.262 250M Will dissolve with heating to 50C, 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 250M Light sensitive
Requires adding OH- to dissolve
AzF 33173-53-4 10% DMSO
90% Water
206.204 10mM 2.062 250M Light sensitive
Requires heating and shaking to dissolve
CNF 167479-78-9 Water 190.2 10mM 1.902 250M Dissolves at room temperature with ~15 minutes of shaking
L-DOPA 59-92-7 Water 197.1879 10mM 1.972 250M Oxidizes quickly and turns black in solution
5HTP 4350-09-8 Water 220.2246 10mM 2.202 250M

FAA: free amino acid
S: salt
HS: hydrated salt

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Topic revision: r49 - 25 Oct 2018 - 22:30:31 - Main.SeanLeonard
 
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