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Stock, conc	Preparation & Storage (Bennett Lab)	Volumes, Tubes	Location
10× T4 ligase buffer	Thawed, precipitates resuspended with vortexing/slight warming.	50 µL aliquots	Rack 1.5, -20°
10 mM dNTPs		20 µL aliquots	Rack 1.5, -20°
Antibiotics
chl^{25 (mg/mL)}	Ethanol, -20° liquid; stable for ≥1 year.	15 mL

Freezer

Fr 1 door
amp¹⁰⁰	40% ethanol, -20° liquid; stable enough for ≥6 mo ⁽⁸⁾. Definitely in water, -20° solid for ≥6 mo^(10,11).	15 mL

Freezer

Fr 1 door
kan⁵⁰	Water, 0° liquid; stable for ≥1 year.	15 mL

Freezer

Fr 0 door
spt⁵⁰	Water, 0° liquid; stable for ≥1 year.	15 mL

Freezer

Fr 0 door
gen¹⁰	Water, -20° solid; stable for ≥1 year.	~1 mL	Rack 1.5
str¹⁰⁰	Water, -20° solid; stable for ≥1 year.	~1 mL	Rack 1.5, -20°
tet¹⁰	Ethanol, -20° liquid; stable in dark for ≥1 year?	~1 mL	Rack 1.5, -20°
zeo¹⁰⁰	?, -20° solid; stable in dark for ≥1 year.	~1 mL	Rack 1.5, -20°

Antibiotics

Antibiotic	Class, Target	Abbr*	Stock Concentration		Solvents, Temp	Notes	Rich Medium
			Relative	m/V			1× c, Multi-Copy; Range		1× c, Single-Copy	Stability
Chloramphenicol	–, ribosome	chl	1000×	25 mg/mL	Ethanol, -20°(l)		25 or 34 mg/L ⁽³⁾	≥25–340 mg/L ⁽⁵⁾	10 mg/L ⁽¹⁾ or 5 mg/L ⁽⁶⁾	All: ≥1 yr ^(8–10)
Ampicillin·Na Carbenicillin·2Na	penicillin, peptidoglycan	amp car	1000×	100 mg/mL	40% ethanol, -20°(l) or water, -20°(s)	40% EtOH keeps it liquid at -20°C. Substitutes carbenicillin.	100 mg/L ⁽³⁾	≥100–200 mg/L	50 mg/L	Stock: ≥6 mo with <¼ loss in water ^{(10, 11)}. Agar: selective for 4 mo ⁽⁸⁾, 44% loss every 4 mo⁽⁹⁾.
Kanamycin A·SO₄	aminoglycoside, ribosome	kan	1000×	50 mg/mL	Water, 0°(l); -20°(s)	Insoluble in 40% ethanol. Precipitates in 35% DMSO.	50 mg/L ⁽³⁾	≥50–500 mg/L ⁽⁵⁾	20 mg/L ⁽¹⁾	All: ≥1 yr ^(8–10)
Spectinomycin·2HCl·5H₂O	aminoglycoside, ribosome	spt	1000×	50 mg/mL	Water, 0°(l); -20°(s)	Precipitates in 35% DMSO.	50 mg/L ⁽³⁾	≥50–500 mg/L ⁽⁵⁾	20 mg/L ⁽¹⁾	All: ≥1 yr ^(8–10)
Streptomycin·SO₄	aminoglycoside, ribosome	str	1000×	100 mg/mL	Water, 0°(l); -20°(s)	.				All: ≥1 yr ^(8–10)
Trimethoprim	diaminopyrimidine, FolA (DHFR)	tmp	1000×	15 mg/mL	DMSO or 1% acetic acid	Insoluble in water or ethanol. Exogenous thymine can lead to evolution of resistance.	15 mg/L?		10 mg/L ⁽²⁾	?
Apramycin·SO₄	aminoglycoside, ribosome	apr	1000×	?	Water	.	?		?	All: ≥1 yr ^(8–10)
Gentamicin C·SO₄	aminoglycoside, ribosome	gen	1000×	10 mg/mL?	Water	.	10 mg/L⁽⁸⁾		10 mg/L ⁽¹⁾	All: ≥1 yr ^(8–10)
Erythromycin	macrolide, ribosome	ery	100×	50 mg/mL	Water, ethanol, DMSO	.	500 mg/L		200 mg/L⁽¹²⁾	?
Tetracycline·HCl	tetracycline, ribosome	tet	1000×	10 mg/mL	Ethanol	Photolabile.	10 mg/L ⁽³⁾	10–15 mg/L	7.5–15 mg/L ^(1,12)	Stock: 4° ≤1 wk, -20° ≤2 mo ⁽¹⁰⁾
Doxycycline	tetracycline, ribosome	dox			Ethanol	Photolabile?
Zeocin (Phleomycin D1·Cu²⁺)	glycopeptide, DNA	zeo	1000×	25/100 mg/mL	Water	Use 25 mg/L with LB-Lennox (5 g/L NaCl), pH 7.5. Or use 100 mg/L for high-salt LB. Light-sensitive. Limit freeze-thaws.	25 mg/L^{(7, low-salt)} 100 mg/L	25–50 mg/L ^{(7, low-salt)}		Agar: 1 mo^(Invivogen)
Hygromycin B	aminoglycoside, ribosome	hyg	1000×	200 mg/mL	Water	.	200 mg/L ⁽³⁾			?

Note: aminoglycoside solutions are generally found to be stable for more than a year in 4° water or agar^(8–10).

* Follow American Society of Microbiology AAC abbreviation guidelines when possible

References

Poteete, Anthony R., Charles Rosadini, and Christine St. Pierre. "Gentamicin and other cassettes for chromosomal gene replacement in Escherichia coli." Biotechniques 41.3 (2006): 261-264. https://doi.org/10.2144/000112242
Kittleson, Joshua T., Sherine Cheung, and J Christopher Anderson. "Rapid optimization of gene dosage in E. coli using DIAL strains." Journal of biological engineering 5.1 (2011): 1-7. https://doi.org/10.1186/1754-1611-5-10
Addgene recommendations https://www.addgene.org/mol-bio-reference/
Qiagen: Growth Of Bacterial Cultures (qiagen.com)
Sara Molinari, unpublished data, p15A with cat, aphA1, aadA.
Marionette Sensor Collection: Meyer, Adam J., et al. "Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors." Nature chemical biology 15.2 (2019): 196-204. https://doi.org/10.1038/s41589-018-0168-3
Zeocin | Thermo Fisher Scientific
Bennett Lab experience, where an antibiotic "working" means it kills typical lab E. coli strains in broth or agar sufficiently to not elicit any concern.
Ryan, Kenneth J., et al. "Stability of antibiotics and chemotherapeutics in agar plates." Applied microbiology 20.3 (1970): 447-451. https://doi.org/10.1128/am.20.3.447-451.1970
Berendsen, B. J. A., I. J. W. Elbers, and A. A. M. Stolker. "Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection." Food Additives & Contaminants: Part A 28.12 (2011): 1657-1666. https://doi.org/10.1080/19440049.2011.604045 Sci-Hub.
Okerman, Lieve, Johan Van Hende, and Lieven De Zutter. "Stability of frozen stock solutions of beta-lactam antibiotics, cephalosporins, tetracyclines and quinolones used in antibiotic residue screening and antibiotic susceptibility testing." Analytica chimica acta 586.1-2 (2007): 284-288. https://doi.org/10.1016/j.aca.2006.10.034
Ferrières, Lionel, et al. "Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery." Journal of bacteriology 192.24 (2010): 6418-6427. https://doi.org/10.1128/JB.00621-10

Lab Improvement Weekly: Investigative Report on Ampicillin. By Shyam Bhakta.

This week's investigative report examines everyone's favorite yet hated bacteriolytic antibiotic: ampicillin.

A 1990 paper¹ discusses how the secreted β-lactamase in a saturated preculture of an Ampᴿ strain can quickly consume all the ampicillin in a subculture. A stationary culture of ampicillin resistant cells can have such a concentration of β-lactamase that even a 1/200 – 1/1000 dilution will still contain enough β-lactamase to consume all the fresh ampicillin before all the non-resistant cells from the stationary phase culture have been killed. Even without any initial β-lactamase, enough β-lactamase is secreted quickly that toxic plasmids can quickly be lost midway through growth without facing selection. The authors recommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid. And before subculturing, cells are best washed for Ampᴿ selection.

Ampicillin's bactericidal activity has a pH optimum is 5.5–6 and is tenfold less at pH 8 ³.

A paper on stability of antibiotics in solid medium over time ² found that 4 wk-old amp plates stored cold have a 30–50% loss in [amp] (causing a 10% change in zone of inhibition size), BUT no change (especially at our high concentration) that would dip below typical Amp^S strain's MIC. Two-month-old plates would seem to be the limit, as after the resulting fourfold reduction in [amp] would approach the MIC. The rest of our common four antibiotics have no significant changes in agar over 2 months.

Activity reduction of a 100 mg/mL (1000×) aqueous, unbuffered solution of ampicillin³ ⁷:

4° 20° -20°

1 d: 15%, 28%

2 d: 33%, 45%

7 d: 65%, 81%

6 mo: <10%

A number of studies have indicated that the stability of ampicillin in solution appears to be a function of pH, temperature, and even the identity of the buffer. Although ampicillin in any form is more readily soluble in base, it rapidly loses activity when stored above pH 7.0. ⁴ ⁵ ⁶ Optimal conditions for storage were suggested as 2–8°C, pH 3.8–5, retaining more than 90% activity for a week. Another review noted that the buffer used can also affect stability: at pH 7, Tris is "highly deleterious to the stability… but not so at pH 5;" citrate is fine at pH 7 but not at pH 5; acetate buffer seems best at pH 6.5 ⁵.

Carbenicillin is anecdotally more chemically stable in media, especially acidic and hot, and more resistant to β-lactamase, but I can only find published evidence that supports the former ⁸; I'm suspicious/doubt the latter claim. Maybe more carb survives 50° agar, but more amp (I've read 200 mg/L) can be used if it matters. While media stability ought to give carbenicillin an edge in selection against amp-sensitive mutants over long culture periods, especially in acidifying media (glucose carbon source), peptide broths like LB alkalify over culture time, and many-day culturing or a requirement of maintenance of 1× concentration in sterile media is rare. I've found 1× amp in LB30/TB liquid is selective for cloning for at least 2 wk in the fridge, longer for regular LB. The remaining amp only needs to exceed the MIC by a fewfold. 100–200 mg/L is 8–16-fold the MIC.

A new paper deletes a small part of the AmpR RBS so it makes less β-lactamase. Lowers how much amp is needed and it reduces β-lactamase accumulation to the level that subcultures of AmpR strains from amp to fresh amp medium preserves selection without quickly destroying all the amp ⁹.

¹ https://www.ncbi.nlm.nih.gov/pubmed/2199796?dopt=Abstract

² https://www.ncbi.nlm.nih.gov/pmc/articles/PMC376956/pdf/applmicro00107-0177.pdf

³ https://www.applichem.com/en/shop/product-detail/as/ampicillin-natriumsalz-ibiochemicai/

⁴ Analytical Profiles of Drug Substances, K. Florey, ed., (Academic Press, NY) Vol. 2, 1-61 (1973).

⁵ Gallelli, Amer. J. Hosp. Pharm., 24, 425-433 (1967)."Stability studies of drugs used in intravenous solutions, part one."

⁶ Lynn, B., Brit. J. Intravenous Therapy, 2, 22 (1981). "The stability and administration of intravenous penicillins."

⁷ https://www.sciencedirect.com/science/article/pii/S0003267006021076?via%3Dihub

⁸ https://sci-hub.tw/https://doi.org/10.1177/106002807000400802

⁹ https://doi.org/10.1021/acssynbio.1c00393