Lysozyme, ampicillin, and kanamycin sulfate were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). FA (content > 99%) was obtained from J&K Scientific Ltd. (Shanghai, China). Methyl ferulate (MFA) (content > 99%) was purchased from ThermoFisher Scientific (Shanghai, China). Commercial xylanase (10,000 U/g) was obtained from Macklin Co., Ltd. (Shanghai, China). Wheat bran was obtained from Auchan Supermarket (Wuxi, China). Other chemicals were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Kanamycin sulfate
Manufactured by Sangon
Sourced in China
Kanamycin sulfate is a broad-spectrum antibiotic used in laboratory settings. It is commonly utilized as a selective agent in molecular biology and microbiology experiments to identify and propagate genetically modified organisms.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Sangon (3 mentions)
Silica gel, by Qingdao Haiyang Chemical (3 mentions)
Glycine, by Sangon (2 mentions)
Primestar max dna polymerase, by Takara Bio (2 mentions)
Chloramphenicol, by Sangon (2 mentions)
Lysozyme, by Sangon (1 mentions)
Lc 20ar, by Shimadzu (1 mentions)
Azithromycin, by Maclin Biochemical Technology (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
Real time system, by Bio-Rad (1 mentions)
Luria bertani medium, by Solarbio (1 mentions)
Imidazole, by Sangon (1 mentions)
Histrap hp 5 ml column, by GE Healthcare (1 mentions)
Nah2po4 2h2o, by Sinopharm (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Sangon (1 mentions)
Bradford reagent, by Sangon (1 mentions)
Sodium hydroxide (naoh), by Sinopharm (1 mentions)
Ethanol, by Sinopharm (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
Na2hpo4 12h2o, by Sinopharm (1 mentions)
9 protocols using kanamycin sulfate
1
Enzymatic Hydrolysis of Wheat Bran
2
Culturing and Transforming Duddingtonia dactyloides
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The D. dactyloides isolate CGMCC3.20198 was cultured from a soil sample from Motuo, Tibet, China, by us. This strain and its mutants were cultured on potato dextrose agar (PDA, BDTM, USA), tryptone glucose (TG, BDTM, USA) agar, and corn meal agar (CMA, BDTM, USA) at 28 °C. For conidiation, they were cultured on CMA plates supplemented with 2 g/L KH2PO4. Water agar plates were used to induce trap formation by introducing Caenorhabditis elegans. For stress assays, they were cultured on PDA with different concentrations of chemical stressors at 28 °C. C. elegans was maintained on nematode growth medium (NGM) agar plates at 23 °C and fed with Escherichia coli OP50. E. coli OP50 was cultured in Luria-Bertani (LB) medium at 37 °C. E. coli DH5α was used for plasmid construction and maintenance, and was cultured in LB at 37 °C. A. tumefaciens strain AGL-1 was used for transforming D. dactyloides and was cultured in LB at 28 °C. Transformants of E. coli DH5α and A. tumefaciens AGL-1 were selected using agar plates supplemented with 100 μg/mL kanamycin sulfate (Sangon Biotech, Shanghai, China). The antibiotic solution was filtered with 0.22 μm membrane before use.
3
Organic Synthesis and Analytical Procedures
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All commercial organic
solvents and salts (Sigma Aldrich, TCI, Adamas, J&K, Energy, etc.)
were used without further purification. Cyclic dipeptides were purchased
from GL Biochem (Shanghai) Ltd. Azithromycin was purchased from Macklin,
and kanamycin sulfate was purchased from Sangon Biotech. Flash chromatography
was performed using a 200–300 or 100–200 mesh silica
gel (Qingdao Haiyang Chemical Co., Ltd.). Preparative HPLC separations
were performed using Shimadzu LC-20AR semipreparative solvent delivery
units and a Shimadzu SPD-20A UV detector equipped with a Shimadzu
C18 column (5.0 μm, 20 × 250 mm) at a flow rate of 10 mL/min.
NMR spectra were acquired using a Bruker 400 MHz, and d6-DMSO (J&K) was used as the solvent during NMR analysis.
Optical density (OD) was recorded on a microplate reader SpectraMax
Plus 384 (Molecular Devices, USA). Fluorescence real-time quantitative
PCR was done on a real-time system (Bio-Rad Laboratories, Hercules,
CA).
solvents and salts (Sigma Aldrich, TCI, Adamas, J&K, Energy, etc.)
were used without further purification. Cyclic dipeptides were purchased
from GL Biochem (Shanghai) Ltd. Azithromycin was purchased from Macklin,
and kanamycin sulfate was purchased from Sangon Biotech. Flash chromatography
was performed using a 200–300 or 100–200 mesh silica
gel (Qingdao Haiyang Chemical Co., Ltd.). Preparative HPLC separations
were performed using Shimadzu LC-20AR semipreparative solvent delivery
units and a Shimadzu SPD-20A UV detector equipped with a Shimadzu
C18 column (5.0 μm, 20 × 250 mm) at a flow rate of 10 mL/min.
NMR spectra were acquired using a Bruker 400 MHz, and d6-DMSO (J&K) was used as the solvent during NMR analysis.
Optical density (OD) was recorded on a microplate reader SpectraMax
Plus 384 (Molecular Devices, USA). Fluorescence real-time quantitative
PCR was done on a real-time system (Bio-Rad Laboratories, Hercules,
CA).
4
Construction of V. mimicus deletion strain
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V. mimicus strain SCCF01 (WT), the highly virulent strain mentioned above, was focused on in this study. Both the plasmid pKD4 (Miaolingbio Co., Ltd., Wuhan, China) with a kanamycin (Kan) cassette flanked by FRT sites and the plasmid pCP20 (Miaolingbio Co., Ltd., Wuhan, China) with FLP recombinase were used to construct the deletion strain [27 (link),28 (link)], and plasmid pKD46 (Miaolingbio Co., Ltd., Wuhan, China) was used to provide λ-RED recombinase [27 (link)]. Plasmid pBAD24 (Biofeng Biotech Co., Ltd., Shanghai, China) was used as an arabinose-inducible expression vector for the crp gene for a complementation experiment [29 (link)]. V. mimicus was routinely cultured in Luria–Bertani (LB) medium (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 28 °C. E. coli DH5α was used for harvesting mutant plasmid and was cultured in LB broth at 37 °C. Dulbecco’s Modified Eagle Medium (DMEM) was used for the growth of channel catfish kidney cells (CCK; Courtesy of Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences). Antibiotics were used at the following concentration: 100 μg/mL of ampicillin and kanamycin sulfate (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). In order to activate the pBAD24, a supplementation of 0.2% arabinose was provided when required.
5
Cloning, Expression, and Purification of Proteins
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The plasmid pET30a(+)-6× His-SC, pET28a(+)-6× His-ST-GFP, and the competent Escherichia coli (E. coli) strain BL21(DE3) were obtained from our laboratory. Na2HPO4·12H2O, NaH2PO4·2H2O, CaCl2, NaCl, HCl, NaOH, and ethanol were purchased from Sinopharm (Beijing, China). EDC, Sulfo-NHS, kanamycin sulfate, agar, isopropyl-β-D-thiogalactopyranoside (IPTG), imidazole, glycine, 2-(N-morpholino)ethanesulfonic acid (MES), BSA, SDS-PAGE preparation kit, Bradford reagent, and carboxyl-SMBs (300 nm in diameter) were purchased from Sangon Biotechnology (Shanghai, China). A 0.1 M phosphate-buffered saline solution (PBS, pH 7.4) was prepared with 29 mg/mL Na2HPO4·12H2O, 2.965 mg/mL NaH2PO4·2H2O, and 8.766 mg/mL NaCl in distilled water, and the pH was adjusted to 7.4 by 1.0 M HCl. PBS (0.02 M) was prepared with 5.8 mg/mL Na2HPO4·12H2O, 0.593 mg/mL NaH2PO4·2H2O, and 29.22 mg/mL NaCl in distilled water, and the pH was adjusted to 7.4 by 1.0 M HCl. HisTrap™ HP 5 mL columns were obtained from GE Healthcare (Pittsburgh, PA, USA). Amicon® Ultra-15 centrifugal filter devices were purchased from Merck Millipore (Darmstadt, Germany).
6
Bacterial Expression System Setup
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Tryptone and yeast extract were purchased from OXOID (Shanghai, China), isopropyl β-D-1-thiogalactopyranoside (IPTG, >99%) and kanamycin sulfate (>99%) were bought from Sangon (Shanghai, China) and steroid compounds were purchased from Aladdin (Shanghai, China). All chemicals were of chemical purity and commercially available. Prime STAR Max DNA polymerase was bought from Takara (Shanghai, China), T5 super PCR Mix (Colony) DNA polymerase, DNA Maker, Trelidf™ Prestained Protein Ladder and TS-GelRed Nucleic acid dye were purchased from TSINGKE (Beijing, China). T5 exonuclease was obtained from New England Biolabs (Beverley, MA).
7
Cholesterol Oxidation Enzymatic Assay
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All solvents and reagents were of the highest purity commercially available, unless noted otherwise. Isopropyl-β-d -thiogalactopyranoside (IPTG), acrylamide, N,N′-methylenebisacrylamide, ammonium persulfate, 30% acrylamide, TEMED, glycine, Tris, DTT, ampicillin sodium, kanamycin sulfate, chloramphenicol, hydroxypropyl NADH, NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, catalase and ammonium sulfate were all purchased from Sangon Biotech (Shanghai, China). Cholesterol, Cholesterol propionate, NADPH, 5-aminolevulinic acid, spinach ferredoxin (spFDX) and spinach ferredoxin reductase (spFDR) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-Cholesten-3-one and methyl-β-cyclodextrin were purchased from J&K Scientific Ltd. (Beijing, China). Taq DNA polymerase, PrimeSTAR HS DNA Polymerase (with GC buffer), T4 DNA ligase, restriction endonucleases Nde I and Xho I, T-A cloning kits and DNA MW markers were purchased from Takara (Dalian, China). Cholesterol sodium sulfate was purchased from Ark Pharm Inc. (Libertyville, IL, USA).
8
Synthesis and Characterization of Gold Nanoparticles
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Protamine sulfate salt, tetrachloroauric (III) acid tetrahydrate ( HAuCl 4 ⋅4H 2 O), andsodium citrate (C 6 H 5 Na 3 O 7 ⋅2H 2 O) were purchased from Sigma-Aldrich (USA). Kanamycin sulfate, tetracycline hydrochloride, p-hydroxy-ampicillin, chloramphenicol, ampicillin and gentamicin sulphate were obtained from Sangon Biotechnology Inc. (Shanghai, China). Kanamycin aptamer (5 ′ -TGG GGG TTG AGG CTA AGC CGA-3 ′ ) and cDNA (5 ′ -TCG GCT TAG CCT CAA-3 ′ ) were synthesized by Sangon Biotechnology Inc. (Shanghai, China). Sodium chloride (NaCl), sodium borohydride (NaBH 4 ), magnesium chloride (MgCl 2 ), glucose, and absolute ethyl alcohol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Exonuclease I was purchased from New England Biolabs (Beijing, China). All other chemicals were of analytical reagent grade or higher and used without further purification. Double distilled water (18.2 MΩ, Pall Cascada) was used throughout the experiments.
9
Aspergillus terreus At-ATA cDNA Cloning
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The At-ATA cDNA from Aspergillus terreus sequence, including the NcoI and XhoI restriction sites, was synthesized by General Biosystems (Chuzhou, China), and the plasmid pET-28a(+) was used for gene cloning and DNA sequencing. All PCR primers were synthesized by Qingke Biology Co., Ltd. (Hangzhou, China). PrimeSTAR® Max DNA polymerase was obtained from Takara Biotechnology (Dalian, China) for the polymerase chain reaction (PCR). Dpn I, Yeast extract and tryptone were obtained from Thermo Fisher Scientific (Shanghai, China). Dimethyl sulfoxide (DMSO), 1-(R)-PEA and pyruvate were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). NaCl, NaH2PO4, Na2HPO4, NaOH, DNA ladder, protein marker, protein loading buffer, kanamycin sulfate, isopropyl-β-d-thiogalactoside (IPTG), Ni-NTA Sefinose (TM) Resin (Settled Resin) kit, SDS-PAGE gel kit, and Modified Bradford Protein Assay Kit were obtained from Sangon (Shanghai, China). E. coli BL21(DE3) Chemically Competent Cell, EasyPure® HiPure Plasmid MaxiPrep Kit, EasyPure® Quick Gel Extraction Kit and EasyPure® PCR Purification Kit were purchased from TransGen Biotech (Beijing, China).
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