Kanamycin

Manufactured by Teknova

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Kanamycin is a broad-spectrum antibiotic commonly used in laboratory settings. It is effective against various Gram-positive and Gram-negative bacteria. Kanamycin functions by inhibiting bacterial protein synthesis, thereby preventing the growth and reproduction of targeted microorganisms.

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Kanamycin LB agar plates (3 citations)

18 protocols using kanamycin

Recombinant PglC Protein Production

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PglC from C. jejuni strain 11168 was cloned into the pET24a vector to insert a C-terminal His₆-tag[37 ] or into the pE-SUMO vector [34 (link)]. P24A variants of both constructs were generated using QuikChange II Site-Directed Mutagenesis (Agilent Technologies, Santa Clara, CA) as described previously [37 ].
PglC-His₆ and SUMO-PglC variants were expressed in BL21-CodonPlus (DE3)-RIL E. coli cells (Agilent Technologies) using the Studier auto-induction method.[44 (link)] Overnight cultures grown in 3 mL MDG media (0.5% (w/v) glucose, 0.25% (w/v) sodium aspartate, 2 mM MgSO₄, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄ and 0.2x trace metal mix (from 1000x stock, Teknova, Hollister, CA)) with kanamycin and chloramphenicol (30 μg/mL each) were used to inoculate 0.5 L auto-induction media (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (v/v) glycerol, 0.05% (w/v) glucose, 0.2% (w/v) α-D-lactose, 2 mM MgSO₄, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, and 5 mM Na₂SO₄, 0.2x trace metal mix) containing kanamycin (90 μg/mL) and chloramphenicol (30 μg/mL). Cells were grown for 4 hr at 37 °C followed by an additional 16-18 hr at 16 °C and then harvested at 3700 × g for 30 min. Cells were stored at −80 ℃ until purification.

Entova S., Guan Z, & Imperiali B. (2019). Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates. Archives of biochemistry and biophysics, 675, 108111.

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Bacterial Growth Conditions

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Bacterial strains and plasmids used in this study are listed in Supplementary Table 3. Bacterial strains were grown in Lysogeny Broth (LB, Difco) or M9 minimal medium supplemented with 0.2% (vol:vol) glucose (M9-glucose) at 37 °C. 0.5% Casamino acids, pantothenate (f.c. 2 µg ml⁻¹), nicotinamide (f.c. 2 µg ml⁻¹), thiamin (f.c. 2 µg ml⁻¹), and biotin (f.c. 100 ng ml⁻¹) were supplemented to M9-glucose for growing S. aureus USA300 strain. When required, media were amended with penicillin G (Sigma-Aldrich) 150 µg ml⁻¹ or kanamycin (Teknova) 50 µg ml⁻¹.

Minato Y., Dawadi S., Kordus S.L., Sivanandam A., Aldrich C.C, & Baughn A.D. (2018). Mutual potentiation drives synergy between trimethoprim and sulfamethoxazole. Nature Communications, 9, 1003.

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Quantifying Bacterial Burden in Murine Meningitis

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CFU assays from blood and CSF of sham and meningitis mice were performed 48 hours after injection. Mice were fixed onto a stereotactic frame in a manner similar to the orientation used for intracisternal injections. To sample the CSF, the injection site was reopened and the dura mater punctured with a glass micropipette to aspirate 5–10 μl CSF. Mice were then removed from the frame and 100 μl of blood was collected by cardiac puncture. For skull and tibia CFUs, bones were aseptically harvested from sham and meningitis groups, and the meninges were dissected from the skull in sterile PBS containing 5% BSA/2 mM EDTA. Pilot experiments were performed to titer dilutions of CSF, blood, skull or tibia homogenate necessary to visualize bacterial growth on blood agar plates containing 50 μg/ml kanamycin (TEKnova Cat#T0194). A cell-spreader was used to evenly distribute homogenates across the plate and the plate was stored in a 37°C for 24 hours prior to analysis. Plates were photographed and colonies quantitated for relative comparisons.

Pulous F.E., Cruz-Hernández J.C., Yang C., Kaya Ζ., Paccalet A., Wojtkiewicz G., Capen D., Brown D., Wu J.W., Schloss M.J., Vinegoni C., Richter D., Yamazoe M., Hulsmans M., Momin N., Grune J., Rohde D., McAlpine C.S., Panizzi P., Weissleder R., Kim D.E., Swirski F.K., Lin C.P., Moskowitz M.A, & Nahrendorf M. (2022). Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis. Nature neuroscience, 25(5), 567-576.

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Curli Protein Expression in E. coli

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EcN Prop-Luc or PBP8 cells were transformed with the corresponding pBbB8k plasmids by electroporation to create variants of curli producing cells and plated onto LB agar plates containing 50 μg mL⁻¹ kanamycin (Teknova) and incubated overnight at 37 °C. Individual colonies were inoculated in 5 mL of LB media containing 50 μg mL⁻¹ kanamycin, and grown overnight in 37 °C shaking incubator. Overnight starter cultures were diluted 100-fold in new LB media containing 50 μg mL⁻¹ kanamycin at desired volumes and incubated while shaking at 37 °C until the refreshed cultures reached a log phase at an optical density (OD) at 600 nm of 0.5 to 0.8. Then, protein expression was induced by adding l-(+)-arabinose to a final concentration of 0.05% (weight per volume). The induced cultures were grown in 37 °C shaking incubator overnight to allow protein expression.

Praveschotinunt P., Duraj-Thatte A.M., Gelfat I., Bahl F., Chou D.B, & Joshi N.S. (2019). Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nature Communications, 10, 5580.

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Cloning and Transforming E. coli

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All constructs were designed with a CMV promoter using Gibson or restriction cloning (see list of constructs) and transformed into chemically competent DH5ɑ E. coli cells (Invitrogen). Transformed E. coli were plated on either 50 μg/mL kanamycin (Teknova, cat #K2151) or 100 μg/mL carbenicillin (Gold Biotechnology, cat #C-103–5) LB agar (Teknova, cat #L9115) plates overnight at 37 °C. Single colonies were picked and grown overnight in 5 mL LB Broth (Alfa Aesar, cat #AAJ75854A1) with kanamycin or carbenicillin at 37 °C with shaking at 220 rpm. The following day, shaking cultures were mini-prepped (Zymo, cat #D4212) and sent for Sanger Sequencing (Azenta). Sequence-verified plasmids were then midi-prepped (Zymo, cat #D4201) for use in transfection.

Black H.H., Hanson J.L., Roberts J.E., Leslie S.N., Campodonico W., Ebmeier C.C., Holling G.A., Tay J.W., Matthews A.M., Ung E., Lau C.I, & Whiteley A.M. (2023). UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS. eLife, 12, e79452.

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Recombinant Protein Expression in E. coli

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Briefly, the target specific vector was transformed into either Top 10 or BL21 (DE3) Escherichia coli cells. A starter culture containing either 100 μg/mL (final concentration) of ampicillin or 50 μg/mL (final concentration) kanamycin (Teknova) was inoculated with a single colony and grown 16 hours at 37°C. This was then transferred to 8 liters of terrific broth (Teknova) containing the appropriate antibiotic as described above, which was grown to OD₆₀₀ = 0.6. Protein expression was induced by adding either 0.1% arabinose or 1mM isopropyl-beta-D-thiogalactopyranoside (IPTG) (VWR) as appropriate and the cultures were grown at either 25°C or 30°C overnight or at 37°C for three hours. The cells were harvested by centrifugation (Beckman) at 5000 rpm for 15 minutes and the pellets were collected and stored at –80°C.

Clifton M.C., Dranow D.M., Leed A., Fulroth B., Fairman J.W., Abendroth J., Atkins K.A., Wallace E., Fan D., Xu G., Ni Z.J., Daniels D., Van Drie J., Wei G., Burgin A.B., Golub T.R., Hubbard B.K, & Serrano-Wu M.H. (2015). A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1. PLoS ONE, 10(4), e0125010.

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Protein Expression and Purification Protocol

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Isopropyl-β-D-thiogalactopyranoside (IPTG), anhydrous sodium phosphate, and dibasic potassium phosphate were purchased from DOT Scientific. The buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycerol, and imidazole were purchased from Fisher Scientific. Kanamycin was purchased from Teknova. Magnesium sulfate heptahydrate, L-β-homolysine and D-Lysine were purchased from Chem-Impex International. Ni(II)-nitrilotriacetic acid agarose (Ni-NTA) resin was purchased from Qiagen. L-Allylglycine was purchased from Cayman Chemical Company. 4,4,5,5-[²H₂]-L-Lysine (d₄-L-Lys) was purchased from Cambridge Isotope Laboratories. L-norleucine was purchased from Alfa Aesar. Trans-4,5-dehydro-DL-Lysine was purchased from Bachem. 6-Hydroxy-L-norleucine was purchased from Acrotein Chembio Inc. 4RS-Cl-DL-Lysine was purchased from Akos GmbH (Steinen, Germany). EZNA Plasmid DNA Mini Kit was purchased from Omega Bio-Tek. Oligonucleotide primers were purchased from Integrated DNA Technologies. Polymerase chain reaction (PCR) reagents, Q5 DNA Polymerase Master Mix, restriction enzymes, restriction enzyme buffers, and T4 DNA ligase were purchased from New England Biolabs. Escherichia coli (Ec) DH5α and Rosetta(DE3) competent cells were purchased from Novagen. ⁵⁷Fe⁰ was purchased from ISOFLEX, USA. All other chemicals were purchased from Millipore Sigma.

McBride M.J., Nair M.A., Sil D., Slater J.W., Neugebauer M., Chang M.C., Boal A.K., Krebs C, & Bollinger JM J.r. (2022). A Substrate-triggered μ-Peroxodiiron(III) Intermediate in the 4-Chloro-L-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate. Biochemistry, 61(8), 689-702.

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Antibiotic Screening and Growth Curve

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Cultures for growth curve and antibiotic screening procedures were started from seed culture diluted to 0.02 OD₆₀₀ or 2.5% (v/v), respectively, and were grown in 10 g/L TSBG. New tubes were opened at each time point to maintain anaerobic conditions throughout and all measurements were made in biological triplicate. Antibiotics used included kanamycin (Teknova, Hollister, CA), tetracycline (Thermo Scientific, Waltham, MA), ampicillin (Research Products International, Mount Prospect, IL), gentamicin (Fisher Scientific, Waltham, MA), spectinomycin (VWR, Radnor, PA), and chloramphenicol (VWR, Radnor, PA) at concentrations listed in Results and Discussion. Growth repression for each sample was calculated using Eq 1. Each sample OD₆₀₀ (G_S) was divided by average growth in wild type (G_WTavg), converted to a percentage and subtracted from 100 to obtain the percent repression. Growth repression was averaged across three sample replicates for each treatment.

Long D.S., Immethun C.M., Vallecilla-Yepez L., Wilkins M.R, & Saha R. (2021). One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z. PLoS ONE, 16(5), e0245407.

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Quantifying Bacterial Burden in Murine Meningitis

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Cloning Protocols with Methylation-Sensitive Sites

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All bacteria were grown in Luria-Bertani (LB) broth (pH 7.5) or on LB agar supplemented with 100 μg/mL of ampicillin (Thermo Fisher Scientific; BP1760-25) or 50 μg/mL of kanamycin (Teknova; K2127) as required. Prior to cloning, all plasmids were transformed into dam/dcm-negative Escherichia coli strain JM110 obtained from Agilent (catalog #200239) due to the presence of methylation-sensitive restriction sites in the cloning protocols. After cloning, plasmids were transformed into E. coli TOP10 bacteria. pUC18 and sterile dH₂O were used as positive and negative controls, respectively.

Pay S.L., Qi X., Willard J.F., Godoy J., Sankhavaram K., Horton R., Mitter S.K., Quigley J.L., Chang L.J., Grant M.B, & Boulton M.E. (2018). Improving the Transduction of Bone Marrow–Derived Cells with an Integrase-Defective Lentiviral Vector. Human Gene Therapy Methods, 29(1), 44-59.

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