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
3′azido-2′,3′-dideoxythymidine	AZT	10 mg/ml	200 µg/ml	50×	ddH₂O	link	Can substitute pharma grade ziduvidine. 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 ug/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 ug/ml	1,000×	ddH₂O	link	Degrades noticeably over the course of 1 week at 4C
Gentamicin	Gen	5 mg/ml	5 µg/ml	1,000×	ddH₂O	link	For non-canonical experiments, use 20µg/mL in culture (preferably from 20mg/mL 1000X stock)
Kanamycin	Kan	50 mg/ml	50 µg/ml	1,000×	ddH₂O	link	Plates lose sensitivity after 2 weeks
Nalidixic Acid	Nal	30 mg/ml	30 µg/ml	1,000×	1 M 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 10 M NaOH	link	Toxic to humans, use PPE!
Spectinomycin	Spec	60 mg/ml	60 µg/ml	1,000×	ddH₂O	link
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
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 4C 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 20C 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.

Antibiotic (Chemical Form)	Abb	MW	Stock Conc.	Volume	Mass	Solvent	Notes
Chloramphenicol	Cam	323.13	20 mg/ml	50 ml	1.00 g	EtOH

Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
docx	Making_stock_antibiotics_and_other_reagents.docx	r3 r2 r1	manage	89.7 K	2014-04-16 - 18:28	CraigBarnhart	How to make stock antibiotic solutions

Barrick Lab > ProtocolList > ProtocolsGeneGorgingMarker > ProtocolsAntibioticStockSolutions

Contributors to this topic

JeffreyBarrick, CraigBarnhart, DanielDeatherage, AurkoDasgupta, SeanLeonard, AlexaMorton, AlvaroRodriguez, JordanMonk, GabrielSuarez, ZuberiAshraf, ElizabethRobinson, TylerDeJong, KateElston, SimonDAlton, DaciaLeon, BrianRenda, DennisMishler, IsaacGifford

Topic revision: r35 - 2015-07-24 - 17:39:05 - Main.DennisMishler