| 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 | |||
| chl25 (mg/mL) | Ethanol, -20° liquid; stable for ≥1 year. | 15 mL | 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 | Fr 1 door |
| kan50 | Water, 0° liquid; stable for ≥1 year. | 15 mL | Fr 0 door |
| spt50 | Water, 0° liquid; stable for ≥1 year. | 15 mL | 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, common E. coli | Common resistance genes | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| m/V | Relative (E. coli) | 1× c, Multi-Copy; Range | 1× c, Single-Copy | Stability | |||||||
| Chloramphenicol | misc, ribosome | chl | 25 mg/mL | 1000× | Ethanol, -20°(l) | ChlR gene is toxic to E. coli in minimal media | 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) | Chloramphenicol acetyltransferase Type A1, cat. |
| Ampicillin·Na Carbenicillin·2Na | penicillin, peptidoglycan | amp | 100 mg/mL | 1000× | 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). | β-lactamase (bla). |
| Kanamycin A ·SO4 | aminoglycoside, | kan | 50 mg/mL | 1000× | 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) | Aminoglycoside 3′-phosphotransferase Type I or II, aphA1 / aph(3')-I / nptI, aph(3')-II / nptII. Type IIIa works in Gram(+). |
| Spectinomycin·2HCl·5H2O | aminoglycoside, ribosome | spt | 50 mg/mL | 1000× | Water, 0°(l); -20°(s) | Precipitates in 35% DMSO. Using 120 mg/L prevents spontaneous genomic resistance evolving (13). | 50 mg/L(3) or 120 mg/L(13) | ≥50–500 mg/L (5) | 20 mg/L (1) | All: ≥1 yr (8–10) | Aminoglycoside 3″-adenylyltransferase, aadA. |
| Streptomycin·SO4 | aminoglycoside, ribosome | str | 100 mg/mL | 1000× | Water, 0°(l); -20°(s) | Genomic resistance via rpsL mutation is common in lab strains. | 100 mg/L | All: ≥1 yr (8–10) | Plasmids: Aminoglycoside 3″-adenylyltransferase, aadA. Genomic resistance usually rpsL mutant. | ||
| Trimethoprim | diaminopyrimidine, FolA (DHFR) | tmp | 15 mg/mL | 1000× | DMSO or | Insoluble in water or ethanol. Exogenous thymine can lead to evolution of resistance. | 15 mg/L? | 10 mg/L (2) | ? | Dihydrofolate reductase type II, dfrB2 . | |
| Apramycin·SO 4 | aminoglycoside, ribosome | apr | ? | 1000× | Water | . | ? | ? | All : ≥1 yr (8–10) | Aminoglycoside N3-acetyltransferase, aac(3′)-IV / aacC4 . | |
| Gentamicin C ·SO 4 | aminoglycoside, ribosome | gen | 10 mg/mL? | 1000× | Water | . | 10 mg/L (8) | 10 mg/L (1) | All : ≥1 yr (8–10) | Aminoglycoside-N3-acetyltransferase, aac(3′)-I / aacC1 . | |
| Erythromycin | macrolide, ribosome | ery | 50 mg/mL | 100× | Water, ethanol, DMSO | . | 500 mg/L | 200 mg/L (12) | ? | 23S rRNA(adenine2085) N6-methyltransferase, ermC . | |
| Tetracycline·HCl | tetracycline, ribosome | tet | 10 mg/mL | 1000× | 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) | metal:tetracycline/H+ antiporter, class C, tetA(C). |
| Doxycycline | tetracycline, ribosome | dox | | Ethanol | Photolabile? | | ? | ||||
| Zeocin (Phleomycin D1·Cu2+) | glycopeptide, DNA | zeo | 25/100 mg/mL | 1000× | 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) | bleomycin/phleomycin binding protein, ble. | |
| Hygromycin B | aminoglycoside, ribosome | hyg | 200 mg/mL | 1000× | Water | . | 50-200 mg/L (3) | ? | Hygromycin B phosphotransferase, hph . | ||
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
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) Note: these stock and working concentrations are uncommon.
- Sara Molinari, unpublished data, p15A with common genes of 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 lab E. coli cloning strains in broth, or in agar without forming a lawn of the non-resistant base strain while selecting for colonies harboring the introduced resistance gene.
- 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
- Barrick Lab wiki: antibiotics
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 time² 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 AmpS 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