Ampicillin

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Ampicillin is a broad-spectrum antibiotic used in laboratory settings. It is a penicillin-based compound effective against a variety of gram-positive and gram-negative bacteria. Ampicillin functions by inhibiting cell wall synthesis, leading to bacterial cell lysis and death.

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Ampicillin sodium salt (74 citations) Ampicillin (Amp, (41 citations) Ampicillin sodium (13 citations) Ampicillin trihydrate (6 citations) Ampicillin (Ap, (4 citations) LB-Ampicillin (3 citations) Ampicillin hydrochloride (2 citations) Ampicillin trisodium salt (2 citations) γ(ampicillin (2 citations) Ampicillin and streptomycin (1 citations)

1 581 protocols using ampicillin

Differentiation of Lgr5+ Progenitors and Spheres

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The flow-sorted cells were diluted to 2 cells/μl in DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), EGF (20 ng/ml; Sigma, E9644), IGF (50 ng/ml, Sigma, I8779), heparan sulfate (20 ng/ml, Sigma, H4777), β-FGF (10 ng/ml, Sigma, F0291), and 0.1% ampicillin (Sigma, A9518-5G) and cultured in Costar ultra-low attachment dishes (Costar, 3599) for 5 days and then passaged to the next generation.
For the differentiation assay, we differentiated both flow-sorted cells and spheres. In the cell-differentiation assay, the flow-sorted Lgr5+ progenitors and Lgr5- SCs were cultured to a density of 50 cells/μl on laminin-coated plates using DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), EGF (20 ng/ml; Sigma, E9644), IGF (50 ng/ml, Sigma, I8779), heparan sulfate (20 ng/ml, Sigma, H4777), β-FGF (10 ng/ml, Sigma, F0291), and 0.1% ampicillin (Sigma, A9518) for 10 days. EdU (10 μM, Invitrogen, C10340) was added during the culture in order to label the dividing cells. In the sphere-differentiation assay, the spheres were plated on laminin-coated four-well dishes and cultured in DMEM/F12 medium with 1% N2 (Invitrogen, 17502-048), 2% B27 (Invitrogen, 17504-044), and 0.1% ampicillin (Sigma, A9518) for 10 days.

Cheng C., Guo L., Lu L., Xu X., Zhang S., Gao J., Waqas M., Zhu C., Chen Y., Zhang X., Xuan C., Gao X., Tang M., Chen F., Shi H., Li H, & Chai R. (2017). Characterization of the Transcriptomes of Lgr5+ Hair Cell Progenitors and Lgr5- Supporting Cells in the Mouse Cochlea. Frontiers in Molecular Neuroscience, 10, 122.

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Optimizing Hyaluronidase Antigen Expression

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The expression of hyaluronidase antigenic fragment was optimized using different Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Merck, Germany) concentrations (0.5 and 1 mM), times of induction (two hours, four hours and overnight), temperature (30ºC and 37ºC), culture media [NB (Merck, Germany) 1.5x without glucose containing 100 mg ampicillin (Merck, Germany), NB (Merck, Germany) 1.5x with glucose (Merck, Germany) containing 100 mg ampicillin (Merck, Germany), Luria-Bertani (LB) broth (Merck, Germany) in one liter including 10 g yeast extract (Merck, Germany), 20 g Bacto tryptone broth (Merck, Germany), 0.2% (mass/v) glucose (Merck, Germany), 10 g NaCl (Roche, Germany), 1 g KCl (Roche, Germany), 0.5 g MgCl₂ (Merck, Germany), 0.5 g CaCl₂ (Merck, Germany), 100 mg ampicillin (Merck, Germany) and LB broth (Merck, Germany), and all the mentioned with the same concentration without glucose (Merck, Germany)] with 0.6-0.8-1 absorbance at 600 nm. The expressed proteins were purified using GST-sepharose column according to the manufacture instruction (Sigma, Germany) (29 ).

Sadoogh Abbasian S., Ghaznavi Rad E., Akbari N., Zolfaghari M.R., pakzad I, & Abtahi H. (2014). Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes. Jundishapur Journal of Microbiology, 8(1), e13653.

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Ampicillin Resistance Assay for S. aureus

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Ampicillin resistance was determined by patching S. aureus isolates onto tryptic soy agar (TSA) with 100 μg/ml Ampicillin (Sigma Aldrich, New Zealand) and determining growth after overnight incubation at 37°C. If no concordant results were obtained within three biological replicates data were labeled as ambiguous (+).

Schulz D., Grumann D., Trübe P., Pritchett-Corning K., Johnson S., Reppschläger K., Gumz J., Sundaramoorthy N., Michalik S., Berg S., van den Brandt J., Fister R., Monecke S., Uy B., Schmidt F., Bröker B.M., Wiles S, & Holtfreter S. (2017). Laboratory Mice Are Frequently Colonized with Staphylococcus aureus and Mount a Systemic Immune Response—Note of Caution for In vivo Infection Experiments. Frontiers in Cellular and Infection Microbiology, 7, 152.

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Microinjection of Sea Urchin Eggs

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Adult Strongylocentrotus purpuratus were collected in La Jolla, CA, and held at 11°C (± 1°C) in flow-through seawater aquaria. Spawning and gamete gathering was performed as described previously (Campanale and Hamdoun, 2012 (link)). For microinjection, eggs were held on protamine sulfate coated delta-T dishes (Bio-ptechs, Butler, PA), and mRNA was injected at 2–5% of egg volume (Lepage and Gache, 2004 (link)). Injected embryos were cultured in filtered seawater (FSW) containing 100 µg/ml ampicillin (Sigma, St Louis, MO) at 15°C (± 0.5°C) for 24 hr, then plucked and cultured to gastrula stage in FSW lacking ampicillin.

Campanale J.P., Gökirmak T., Espinoza J.A., Oulhen N., Wessel G.M, & Hamdoun A. (2014). Migration of Sea Urchin Primordial Germ Cells. Developmental dynamics : an official publication of the American Association of Anatomists, 243(7), 917-927.

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Cultivation of Streptococcus gordonii Strains

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The S. gordonii strains used in this study were DL1 (Challis strain; wild type) and its derivative CM100 (DL1 ⓧhsa cat) [6 (link), 7 (link)]. Streptococci were cultured overnight at 37°C in brain heart infusion broth (Becton Dickinson, Franklin Lakes, NJ). The medium was supplemented (as needed) with 200 μg/ml of spectinomycin dihydrochloride or 8 μg/ml of chloramphenicol (Sigma-Aldrich, St. Louis, MO). Escherichia coli BL21 harboring the plasmids with ampicillin resistance was grown in Luria-Bertani broth containing 100 μg/ml ampicillin (Sigma-Aldrich).

Urano-Tashiro Y., Takahashi Y., Oguchi R, & Konishi K. (2016). Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors. PLoS ONE, 11(4), e0154098.

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Aging Human Skeletal Muscle Cell Culture

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Human skeletal muscle cells from an 83‐year‐old donor (HuSkMCs; Cook Myosite Inc, PA 15238, USA) were cultured for six days at 37 °C, 5% CO₂ in growth medium consisting of Myotonic basal medium (Cook Myosite Inc, PA 15238, USA) supplemented with Myotonic growth supplement (Cook Myosite Inc, PA 15238, USA), 20% FCS (Thermo Fischer Scientific), 100 μg/mL ampicillin (Sigma Aldrich), and 10 μg/mL Insulin (Amimed). Cells were then seeded in collagen‐coated 96‐well plates (25 000 cells/well) and in six‐well plates (500 000 cells/well). After 24 h, cells were washed once with differentiation medium consisting of Myotonic differentiation medium (Cook Myosite Inc, PA 15238, USA) supplemented with 1% FCS (Thermo Fischer Scientific) and 100 μg/mL ampicillin (Sigma Aldrich). Cells were starved for 3 h at 37 °C in differentiation medium. Subsequently, HuSkMCs were treated with 1 ng/mL TNFα (R&D system) for different time frames (30 min, 1 h, 3 h, 24 h, 48 h, and 72 h). At the end of incubation, all the plates were washed with phosphate‐buffered saline (PBS) and further processed for the different assays described below.

Ebhardt H.A., Degen S., Tadini V., Schilb A., Johns N., Greig C.A., Fearon K.C., Aebersold R, & Jacobi C. (2017). Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia: a pilot study. Journal of Cachexia, Sarcopenia and Muscle, 8(4), 567-582.

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Preparation and Storage of RNAi Library Clones

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Plates from Ahringer’s RNAi library containing the clones of interest (purchased from the Medical Research Council (MRC) GeneService) were replicated to Luria-Bertani (LB) agar supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich) using a pin replicator (Boekel) and grown overnight at 37 °C. The day after, the selected clones were inoculated in 1.3 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich), tetracycline (15 μg/ml, Sigma-Aldrich) and 8% glycerol in deep well plates (VWR) and incubated overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R incubator shaker). The last column of the plates was left free for convenient control. The next day, 120 μl of the culture was transferred to microtitre plates using a Precision XS Microplate Sample Processor (Biotek, Winooski, VT, USA) and frozen at −80 °C.

Hernando-Rodríguez B., Erinjeri A.P., Rodríguez-Palero M.J., Millar V., González-Hernández S., Olmedo M., Schulze B., Baumeister R., Muñoz M.J., Askjaer P, & Artal-Sanz M. (2018). Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans. BMC Biology, 16, 36.

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Antibacterial Activity of Bioactive Glass

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Experiments of bactericidal activity have been performed on TOP10 chemically competent Escherichia coli (E. coli, Thermo Fisher Scientific, Waltham, MA, USA). Bacteria were previously transformed and carried a plasmid containing ampicillin-resistance cassette. Therefore, bacteria were resistant to ampicillin, facilitating the handling in non-sterile conditions. By using ampicillin in both culture media and plates, contamination by environmental bacteria is prevented. E. coli were grown at 37 °C in Luria-Bertani medium (LB medium, Sigma, St. Louis, MO, USA) in presence of ampicillin (100 μg/mL, Sigma, St. Louis, MO, USA) and constantly shaken at 180 RPM.
Two experimental compositions namely ABG3 and ABG4 were sterilized by autoclaving and subsequently added to sterile LB medium (with 100 μg/mL of ampicillin). For comparative purposes the unmodified bioactive glass BG was also tested. Two experiments were performed to investigate the antibacterial activity of the samples.

Gonzalo-Juan I., Xie F., Becker M., Tulyaganov D.U., Ionescu E., Lauterbach S., De Angelis Rigotti F., Fischer A, & Riedel R. (2020). Synthesis of Silver Modified Bioactive Glassy Materials with Antibacterial Properties via Facile and Low-Temperature Route. Materials, 13(22), 5115.

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Antibiotics Loaded on Modified Microcapillaries

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MCF was modified with PVOH as described above. To load antibiotics onto the internal surface of the PVOH modified MCF, individual microcapillaries of a length of PVOH modified MCF (up to 5 m) were filled using a 30G needle with freshly prepared filter sterilized antibiotic solutions of gentamicin, tetracycline, trimethoprim, ciprofloxacin, ampicillin, amoxicillin and cefotaxime (Sigma Aldrich, UK) at the following concentrations and solvents: gentamicin 5 mg mL⁻¹ in ultrapure water; tetracycline 5 mg mL⁻¹ in water; trimethoprim 3 mg mL⁻¹ in DMF; ciprofloxacin 3 mg mL⁻¹ in 1% HCl; ampicillin 10 mg mL⁻¹ in water; amoxicillin 15 mg mL⁻¹ water; and cefotaxime 4.8 mg mL⁻¹ in water. After 5 minutes of incubation, the excess solution was removed by attaching MCF lengths to a vacuum manifold and dried for 20 minutes per 1 m length, leaving behind a thin film of antibiotic as described previously.^{42 (link)} All antibiotics were loaded at concentrations that would lead to release of the breakpoint concentration into the sample (Table S-1†) based on previously determined loading efficiency quantified by LC-MS and confirmed by phenotypic AST experiments using this method.

Needs S.H., Pivetal J., Hayward J., Kidd S.P., Lam H., Diep T., Gill K., Woodward M., Reis N.M, & Edwards A.D. (2023). Moving microcapillary antibiotic susceptibility testing (mcAST) towards the clinic: unravelling kinetics of detection of uropathogenic E. coli, mass-manufacturing and usability for detection of urinary tract infections in human urine. Sensors & Diagnostics, 2(3), 736-750.

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Expression of Recombinant Osu1 Protein

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The recombinant plasmid pQE30/Osu1, sequence confirmed, was used to transform E. coli SHuffle cells for testing expression. Briefly, the transformation procedure was as follows: 50 ng of pQE30/Osu1 plasmid was blended with 100 µl of competent E. coli/SHuffle cells and maintained in ice for 30 min, then heated the mix for 1 min at 42°C, followed by cooling in ice for 5 min. Afterward, we added 220 µl of Super Optimal broth with Catabolite repression (SOC) medium, and the mix was kept for 60 min at 37°C. After incubation, 50 µl of the mixture were spread over Petri dishes containing (Luria-Bertani) LB agar-media with ampicillin (100 µg/ml) (Sigma, St. Louis, MO, United States). Grown colonies that harbored the pQE30/Osu1 vector were used to screen their expression. Colonies individually were selected, and seeding each in 3 ml of LB broth, including ampicillin plus 1 mM isopropyl ß-D-thiogalactoside (IPTG, Sigma, St. Louis, MO, United States). Then they were incubated overnight in a shaker at 250 rpm and 37°C. Expression was tested qualitatively by SDS-PAGE. Lastly, a positive clone was selected to evaluate the expression of Osu1.

Alvarado D., Cardoso-Arenas S., Corrales-García L.L., Clement H., Arenas I., Montero-Dominguez P.A., Olamendi-Portugal T., Zamudio F., Csoti A., Borrego J., Panyi G., Papp F, & Corzo G. (2021). A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5. Frontiers in Pharmacology, 11, 563858.

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