Transgenic tobacco plants were generated through the agrobacterium-mediated transformation method as previously described (Horsch et al. 1989 ). Fully expanded unstressed leaves from a sterile cultured Nicotiana tabacum cv.Xanthi plant were cut into squares with diameter of 0.5–1.0 cm, and the leaf disks were incubated with LBA4404 agrobacterium harboring a plant expression vector for 10 min. After the inoculation, the disks were placed with their adaxial surfaces facing downwards on co-cultivation medium [3% sucrose, 4.4 g/L MS salt (Duchefa, Netherlands), 2 mg/L 2,4-D (Duchefa, Netherlands), and 200 μM acetosyringone (MBcell, Korea), pH 5.8]. After 2 days of co-cultivation, the disks were transferred into shoot induction medium [3% sucrose, 4.4 g/L MS salt, 50 mg/L kanamycin (Biosesang, Korea), 0.01 mg/L NAA (Duchefa, Netherlands), 0.1 mg/L GA3 (Duchefa, Netherlands), 1 mg/L zeatin (Duchefa, Netherlands), and 250 mg/L cefotaxime (Biosesang, Korea), pH 5.8]; the explants were transferred into fresh shoot induction medium every 2–3 weeks. The regenerated shoots were transferred to root induction medium (3% sucrose, 4.4 g/L MS salt, 50 mg/L kanamycin, and 300 mg/L cefotaxime, pH 5.8). The regenerated plantlets were transferred to soils.
Kanamycin
Manufactured by Biosesang
Sourced in United States
Kanamycin is an antibiotic used in laboratory settings. It is a broad-spectrum aminoglycoside antibiotic that inhibits bacterial protein synthesis, making it effective against a variety of Gram-positive and Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Zeatin, by Duchefa Biochemie (1 mentions)
Ms salt, by Duchefa Biochemie (1 mentions)
2 n morpholino ethanesulfonic acid mes, by Merck Group (1 mentions)
Gwiz luciferase, by Aldevron (1 mentions)
Luria bertani media, by BD (1 mentions)
3 hydroxytyramine hydrochloride, by Merck Group (1 mentions)
Peptone, by Merck Group (1 mentions)
Acetosyringone, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Rifampicin, by Duchefa Biochemie (1 mentions)
3 protocols using kanamycin
1
Generating Transgenic Tobacco Plants
2
Plasmid Transfection and Protein Expression
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We purchased miR-145 from Genolution Pharmaceuticals (Seoul, Korea). We transformed plasmid gWiz-luciferase (Aldevron, USA) into the competent cells, Escherichia coli DH5α, using the heat shock method. gWiz-luciferase was then propagated in bacterial cultures grown in Luria-Bertani media (Becton and Dickinson company, Franklin Lakes, NJ, USA) containing 100 µg/mL of kanamycin (Biosesang Inc., Sungnam, Korea), and purified using a mini DNA-spin kit (iNtRON Biotechnology, Seongnam, Korea). We purchased an antibody for c-Myc and green fluorescent protein (GFP) from Genolution Pharmaceuticals (Seoul, Korea). We obtained branched PEI-linked ssPEI from Pohang University of Science and Technology. We purchased HA from Lifecore Biomedical (Chaska, MN, USA). We purchased 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide from TCI (Tokyo, Japan). Finally, we purchased 3-hydroxytyramine hydrochloride and 2-(N-Morpholino) ethanesulfonic acid (MES) from Sigma Aldrich (St. Louis, MO, USA) and used them without further purification.
3
Agrobacterium-mediated Transient Expression of CRISPR sgRNAs
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The four plasmids harboring different sgRNAs were transformed into Agrobacterium tumefaciens strain GV3101. The transformed GV3101 were incubated in yeast extract peptone (YEP) liquid media [10 g/l of yeast extract (Sigma-Aldrich, USA), 10 g/l of peptone (Sigma-Aldrich, USA), and 5 g/l of NaCl (Sigma-Aldrich, USA), pH 7.2] supplemented with 50 mg/l rifampicin (Duchefa, Netherlands) and 50 mg/l kanamycin (Biosesang, Korea) for overnight at 28°C. In total, 1 ml of overnight culture was transferred into 20 ml fresh YEP media supplemented with 50 mg/l rifampicin and 50 mg/l kanamycin and incubated for 16 h at 28°C. The bacterial solution was centrifuged at 3,500 rpm for 20 min, and the resultant bacterial pellet was resuspended in 10 mM MgCl2 (Sigma-Aldrich, USA) solution supplemented with 200 μM acetosyringone (Sigma-Aldrich, USA) (final OD600 = 0.8–0.9) and incubated at room temperature for 3 h. The bacterial solution was infiltrated into tomato cotyledons using a needless syringe. The ~50 infiltrated cotyledons were harvested 5 days after infiltration for genomic DNA extraction and further PCR analysis. The efficiency of four sgRNAs was evaluated by sequencing analysis of TA-cloned PCR products.
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