We used commercial reagents and solvents without further purification. We also performed all reactions in the air atmosphere unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) performed on Merck TLC Silica gel plates (60 F254), using a UV light for visualization and basic aqueous potassium permanganate or iodine fumes as a developing agent. 1H and 13C NMR spectra were recorded on Bruker Avance 400 instrument with operating frequency of 400 and 100 MHz, respectively, and calibrated using residual undeuterated chloroform (δH = 7.28 ppm) and CDCl3 (δC = 77.16 ppm) or undeuterated dimethyl sulfoxide (DMSO) (δH = 2.50 ppm) and DMSO-d6 (δC = 39.51 ppm) as internal references. The following abbreviations are used to set multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. The purity of the final compounds was checked by liquid chromatography-mass spectrometry (LCMS) in a Shimadzu LCMS-2010A using three types of detection systems such as EDAD, ELSD, and UV and was found to be at least 95%.
Tlc silica gel plates 60 f254
Manufactured by Merck Group
Sourced in Germany
TLC silica gel plates 60 F254 are thin-layer chromatography (TLC) plates made with silica gel as the stationary phase. The plates have a fluorescent indicator F254 that allows for the visualization of separated compounds under UV light. These plates are commonly used in analytical chemistry and organic synthesis for the separation, identification, and purification of various chemical compounds.
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4 protocols using tlc silica gel plates 60 f254
1
Purification and Characterization of Compounds
2
Spectroscopic Characterization of Synthesized Compounds
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Melting points were detected by the Tomas–Hoover capillary melting apparatus without any correction. All solvents, chemicals, and reagents were obtained from Aldrich Chemical Company (Milwaukee, WI), and El Nasr pharmaceutical chemical companies, Cairo, Egypt. Infrared (IR) spectra were obtained using films on KBr diks on a Schimadzu FT-IR 8400S spectrophotometer and values were presented as cm−1. The purity of the synthesized compounds and the reaction progress was monitored using precoated thin layer chromatography (TLC) silica gel plates 60F254 with a thickness of 0.25 supplied from MERCK, Darmstadt, Germany. The UV lamp was used to monitor the reaction process. 13C NMR and 1H NMR spectra were measured on a Bruker Avance III 400 MHz spectrophotometer (faculty of pharmacy, Benisuef University and Mansoura University, Egypt) using dimethyl sulfoxide (DMSO-d6) or D2O as a solvent. The chemical shift was estimated in ppm on δ scale and J (coupling constant) was estimated in Hertz. Microanalysis for C, H, and N were performed on a PerkinElmer 2400 analyzer (PerkinElmer, Norwalk, CT. USA) at the regional center for mycology and Biotechnology, Al-Azhar University, Egypt. All results were within ± 0.4% of the theoretical values.
3
Chitosan Degradation by Marine Bacterium Y82
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Chitosan (viscosity: 200 mPa·s; deacetylation degree: 95%) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Standard chitosan trisaccharide, disaccharide, and monosaccharide were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The TLC silica gel plates 60 F254 were bought from Merck KGaA (Darmstadt, Germany).
The marine bacterium strain Renibacterium sp. Y82 was isolated from brown seaweed in the Yellow Sea, China. In brief, the chips of rotten brown seaweed were put into the chitosan sole-carbon medium. After culture in the flask for microorganism enrichment and spread plate cultivation for isolation, a Renibacterium sp. strain named Y82 was found to grow in the chitosan sole-carbon medium, signifying the ability to degrade and apply chitosan (detailed data not shown), and stored in the laboratory. E. coli DH5α and BL21 (DE3) were used for plasmid construction and csnY gene expression, respectively. Both these strains were cultured at 37 °C in Luria-Bertani (LB) broth or solid medium with 2% (w/v) agar, into which 50 μg/mL kanamycin was supplemented if necessary. Expression vector pET-28a (+) was purchased from Novagen (Madison, WI, USA).
The marine bacterium strain Renibacterium sp. Y82 was isolated from brown seaweed in the Yellow Sea, China. In brief, the chips of rotten brown seaweed were put into the chitosan sole-carbon medium. After culture in the flask for microorganism enrichment and spread plate cultivation for isolation, a Renibacterium sp. strain named Y82 was found to grow in the chitosan sole-carbon medium, signifying the ability to degrade and apply chitosan (detailed data not shown), and stored in the laboratory. E. coli DH5α and BL21 (DE3) were used for plasmid construction and csnY gene expression, respectively. Both these strains were cultured at 37 °C in Luria-Bertani (LB) broth or solid medium with 2% (w/v) agar, into which 50 μg/mL kanamycin was supplemented if necessary. Expression vector pET-28a (+) was purchased from Novagen (Madison, WI, USA).
4
Chlorophyll Extraction and Quantification from Leaf Tissue
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The chlorophyll content was measured in 20–60 mg of leaf tissue from 32 day-old plants. The material was frozen and ground in liquid nitrogen and chlorophyll was extracted by addition of 1 ml 80% (v/v) acetone and an incubation for 30 min on ice in the dark. The extract was cleared by centrifugation and extraction was repeated twice with 0.5 ml 80% (v/v) acetone without incubation. All supernatants were mixed and used for measurements. The chlorophyll concentration per fresh weight was calculated as described previously [20] (link).
Leaf pigment composition was analyzed by thin-layer chromatography of 500 mg 14 (Col-0 wild type) or 21 day-old seedlings (amiR-SSU1-B). Plant material was frozen, ground in liquid nitrogen and extracted by addition of 0.5 ml 80% (v/v) acetone. The extract was cleared by centrifugation and the extraction was repeated by addition of 1 ml 80% (v/v) acetone and incubation for 30 min on ice in darkness. After clarification 40 µl of the mixed extracts was separated on hydrophobic TLC silica gel plates 60 F254 (Merck KGaA) with methanol:acetone:water (15∶5:1) as mobile phase.
Quantification of PCR products was done using AIDA Image Analyzer v3.12 according to instructions given by the manufacturer (Raytest, Germany).
Leaf pigment composition was analyzed by thin-layer chromatography of 500 mg 14 (Col-0 wild type) or 21 day-old seedlings (amiR-SSU1-B). Plant material was frozen, ground in liquid nitrogen and extracted by addition of 0.5 ml 80% (v/v) acetone. The extract was cleared by centrifugation and the extraction was repeated by addition of 1 ml 80% (v/v) acetone and incubation for 30 min on ice in darkness. After clarification 40 µl of the mixed extracts was separated on hydrophobic TLC silica gel plates 60 F254 (Merck KGaA) with methanol:acetone:water (15∶5:1) as mobile phase.
Quantification of PCR products was done using AIDA Image Analyzer v3.12 according to instructions given by the manufacturer (Raytest, Germany).
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