Genesys 20 spectrophotometer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Genesys 20 spectrophotometer is a compact, easy-to-use instrument designed for basic absorbance measurements in the visible wavelength range. It features a wavelength range of 325 to 1100 nanometers and can perform quantitative and qualitative analyses of various samples.

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Genesys 20 (154 citations) GENESYS 20 Visible Spectrophotometer (16 citations) Spectronic 20 Genesys spectrophotometer (5 citations) GENESYS 20 UV–VIS spectrophotometer (5 citations) Spectrophotometer Genesys (2 citations) ThermoScientific Genesys 20 spectrophotometer (2 citations)

67 protocols using genesys 20 spectrophotometer

Cyanobacterial Circadian Rhythm Assay

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Cyanobacterial strains AMC541 and AMC704 were pre-cultured for two days
at 30 °C with shaking in glass test tubes, then standardized to a density
of OD₇₅₀ = 0.2 with a Genesys 20 spectrophotometer (Thermo Fisher).
Luciferase assays were prepared in 96-well plates (Tecan) (Paddock et al., 2013 (link)). Duplicate plates were
initially entrained in opposite 12 h:12 h LD cycles in separate incubators with
programmed lighting, then transferred to a Tecan Spark plate reader for
measuring bioluminescence (Tecan). Plates were illuminated by white LED strip
lights at 35 μmol photons m⁻²s⁻¹ with continuous light, or 70 μmol
photons m⁻²s⁻¹ in LD cycles of 45′:45′ or
12 h:12 h. Bioluminescence from samples was measured every 90 min for 7 days.
Values from edge wells were discarded due to desiccation risk (Paddock et al., 2013 (link)) and bioluminescence
measurements were analyzed using BioDare2 software (Zielinski et al., 2014 (link)). Data were subjected to
linear detrending and period length was calculated using FFT NLLS (Zielinski et al., 2014 (link)). Due to an unknown
instrument error, in Fig. 2B every 16th
data point was abnormally high, and was removed from the plot; the original
plots are provided in Supplemental file, Fig. S1.

Bishé B., Golden S.S, & Golden J.W. (2022). Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit. Life sciences in space research, 36, 18-26.

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Standardized Phytochemical Characterization

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The standard compounds and reagents (all of analytical grade): MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DMSO (dimethyl sulfoxide), gallic acid, Na₂CO₃, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), Folin-Ciocalteu’s reagent, resazurin, tetracycline, chloramphenicol, kanamycin, acetonitrile (LC-MS grade, gradient grade), formic acid, 4-methoxybenzoic acid, 4-hydroxy-3-methoxycinnamaldehyde, caffeic acid, quercetin, luteolin and pinostrobin PBS were purchased from Merck (Darmstadt, Germany). The standards of 4-hydroxybenzoic acid, p-coumaric acid, ferulic, isoferulic acid, pinobanksin, chrysin, sakuranetin, naringenin, apigenin, kaempferol, isorhamnetin, acacetin and pinocembrin were obtained from Extrasynthese (Genay, France) and galangin from Alfa Aesar, (Haverhill, MA, USA). The antibiotics: ampicillin, oxacillin, teicoplanin, gentamicin, amikacin, fusidic acid, erythromycin, mupirocin, rifampicin, levofloxacin, norfloxacin, linezolid, bacitracin were purchased from Argenda (Poland). Methanol, ethanol, vanillin, benzoic acid, cinnamic acid were purchased from (POCH, Gliwice, Poland). Ultrapure H₂O (18.0 MΩ) was obtained with a Milli-Q Advantage A10 system (Millipore, Billerica, MA, USA). The absorbance of the reaction mixture in Folin-Ciocalteu, DPPH assays were measured using a Genesys 20 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Grecka K., Kuś P.M., Okińczyc P., Worobo R.W., Walkusz J, & Szweda P. (2019). The Anti-Staphylococcal Potential of Ethanolic Polish Propolis Extracts. Molecules, 24(9), 1732.

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DPPH Free Radical Scavenging Assay

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The antioxidant capacity was determined through the free radical scavenging method according to Brand-Williams [38 (link)] including some adaptations. The assay involved the reaction of 0.01 mL of extract dissolution with 0.99 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) dissolution for 30 min at room temperature in the dark; then the absorbance was measured at 517 nm using a Thermo Fisher Scientific^® Genesys 20 spectrophotometer (Thermo Scientific, Rochester, NY, USA). The decrease of absorbance in respect to a DPPH reference (without antioxidant added) is related to the radical-reducing capacity of the extract. The results were expressed as a percentage of inhibition, %Inh, as in Equation (1), where A_sample is DPPH absorbance after addition of an antioxidant, A_blank is the absorbance of blank solution from the sample and A_reference is the original absorbance of DPPH dissolution. Additionally, they are expressed in µmol of Trolox^® equivalent/g of dried extract (TEAC) through a calibration curve. The entire assay was performed with one blank solution and four replicate samples.

Sagaste C.A., Montero G., Coronado M.A., Ayala J.R., León J.Á., García C., Rojano B.A., Rosales S, & Montes D.G. (2019). Creosote Bush (Larrea tridentata) Extract Assessment as a Green Antioxidant for Biodiesel. Molecules, 24(9), 1786.

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Anaerobic Cultivation of E. lenta

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Cells were cultured in hungate tubes and all experiments were performed anaerobically. E. lenta strains were inoculated from single colonies into 10 mL of BHI liquid medium and grown for 48–72 hr at 37°C to provide turbid starter cultures. These were diluted 1:100 in triplicate into 5 mL of basal medium containing 10 mM acetate and either 1 mM dopamine (in water) or vehicle (water). If applicable. molybdate (0.5 mM), tungstate (0.5 mM), DMSO (14 mM), or nitrate (1 mM) were added at the time of inoculation. Cultures were grown anaerobically for 36–72 hr at 37°C. Endpoint growth was assessed by measuring the optical density at 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific). Catechol dehydroxylation was assessed at the end of growth in culture supernatants using the colorimetric method.

Maini Rekdal V., Nol Bernadino P., Luescher M.U., Kiamehr S., Le C., Bisanz J.E., Turnbaugh P.J., Bess E.N, & Balskus E.P. (2020). A widely distributed metalloenzyme class enables gut microbial metabolism of host- and diet-derived catechols. eLife, 9, e50845.

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Microbial Biomass and Lipid Analysis

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Bacterial biomass and carotenoids were analyzed according to Saejung and Chanthakhot [25 ]. Carotenoids were extracted by immersing the cell pellets in methanol-acetone (2:3 v/v) solution overnight until the colorless cells were obtained. The pigment extract was read at 480 and 770 nm using a Genesys 20 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Microbial lipids were extracted from cells by centrifugation of the culture broth at 9000 rpm 4 °C for 15 min. The pellets were resuspended in distilled water after being washed twice with 0.9% NaCl. The pellets were boiled for 10 min in 1 N NaOH, and the cell debris was discarded [27 (link)]. The supernatant was used to examine microbial lipids by saponification with 1.5 M KOH in 80% ethanol following Kwon and Rhee [28 ].

Saejung C., Lomthaisong K, & Kotthale P. (2023). Alternative microbial-based functional ingredient source for lycopene, beta-carotene, and polyunsaturated fatty acids. Heliyon, 9(3), e13828.

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E. coli K-12 NCM3722 Growth Protocol

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E. coli K-12 NCM3722^{67 –69 (link)} was grown in Neidhart’s MOPS minimal media^{70 (link)} with glucose and NH₄Cl as the carbon and nitrogen sources. NCM3722 harboring km::T:_ptet:mCherry, sp:P_con-TetR-LacIq(attB) (NMK104) was used for measurement of mCherry expression under the microscope. NMK80, a NCM3722 harboring P_tet-lacZ (TetR is constitutively expressed in this strain) was used for β-galactosidase assay^{71 (link)}. To prepare experimental cultures, cells were taken from −80 °C stocks and streaked on a LB plate. Single colony was inoculated in 2 mL LB medium and grown at 37 °C with constant agitation at 250 rpm in a water bath (New Brunswick Scientific). To monitor growth, the optical density (OD₆₀₀) of the culture was measured using a Genesys20 spectrophotometer (Thermo-Fisher) with a standard cuvette (16.100-Q-10/Z8.5, Starna Cells Inc). Before cells entered stationary phase, cells were inoculated into 5 mL MOPS minimal medium at very low densities (typically lower than the OD₆₀₀ of ~0.0001) and cultured them overnight (pre-culture). Next morning, the pre-culture was diluted in pre-warmed, 5 mL MOPS minimal medium (experimental culture) to the OD₆₀₀ of ~0.01 and allowed to grow exponentially to desired OD₆₀₀.

Le D., Akiyama T., Weiss D, & Kim M. (2023). Dissociation kinetics of small-molecule inhibitors in Escherichia coli is coupled to physiological state of cells. Communications Biology, 6, 223.

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Peptidoglycan Purification and Enzymatic Digestion

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To purify peptidoglycan free of AtlA [22 (link),37 (link)], OG1RF ΔatlA cells were grown to an OD₆₅₀ of 0.7. The bacteria were centrifuged and peptidoglycan was extracted by treating the pellet with 14 mL 4% sodium dodecyl sulfate (SDS) for 30 minutes at 100°C. Peptidoglycan was washed five times with 20 mL of water and was then incubated with Pronase (200 μg/mL) in 1 mL of Tris-HCl (10 mM) and with trypsin (200 μg/mL) in 1 mL of phosphate buffer overnight at 37°C. Peptidoglycan was washed two times with 20 mL of water. To digest peptidoglycan with recombinant protein, 10 μg/mL of either recombinant AtlA or AtlA’ was added to purified peptidoglycan. Turbidity at 450 nm was measured every 30 minutes on a Genesys 20 Spectrophotometer (Thermo Fisher) over a 6-hour time period at 37°C in 25 mM Tris-HCl pH 7.5, 100 mM NaCl.

Stinemetz E.K., Gao P., Pinkston K.L., Montealegre M.C., Murray B.E, & Harvey B.R. (2017). Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation. PLoS ONE, 12(10), e0186706.

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Quantitative Analysis of Aucubin

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Quantitative analysis of aucubin was performed according to Polish Pharmacopoeia VI [146 ] and literature data [151 ].
Aucubin quantitative analysis was performed with the use of the spectrophotometric method. To build the calibration curve, aucubin stock solutions were prepared with the following concentrations: 0.015 mg/mL, 0.0075 mg/mL, and 0.00375 mg/mL). An amount of 0.5 mL from each solution was transferred to three test tubes. Then, 1 mL of methanol, 2 mL of a 4-dimethylaminebenzaldehyde solution containing 0.5 mol/L of hydrochloric acid (Ehrlich reagent), and 1.5 mL of distilled water were added to the test tube. The mixture was heated for 3 min in a boiling water bath (EkoTerm TW 20, Julabo, Schwalbach, Germany). Next, the test tube contents were transferred to a 10-mL flask and diluted to the volume with distilled water. After 15 min, the absorbance of the blue solutions was measured using a Genesys 20 spectrophotometer (Thermo Fisher Scientific, Spectronic, Texas City, TX, USA) at 590 nm as the analytical wavelength. The experiments were triplicated. The average results were applied to prepare the calibration curve (Figure 13).

Konarska A., Weryszko-Chmielewska E., Matysik-Woźniak A., Sulborska A., Polak B., Dmitruk M., Piotrowska-Weryszko K., Stefańczyk B, & Rejdak R. (2021). Histochemical and Phytochemical Analysis of Lamium album subsp. album L. Corolla: Essential Oil, Triterpenes, and Iridoids. Molecules, 26(14), 4166.

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Bacterial Cell Culture and Plasmid Extraction

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Library cell stocks containing mixtures of pJK-series eGFP expression plasmids were thawed on ice and washed with M9 minimal media [22 (link)]. Cultures were inoculated to an OD₆₀₀ of 0.03 in M9 minimal media supplemented with 4 g/L glucose and carbenicillin (50 μg/mL). Cultures were grown at 37°C and 250 rpm to an OD₆₀₀ of 0.6. Cell growth was monitored every 45 minutes by OD₆₀₀ measured on a Genesys 20 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Cells were washed with M9 media. The cells were used to re-inoculate 2.5 mL of fresh media to an OD₆₀₀ of 0.03. Cultures were again grown to an OD₆₀₀ of 0.6 (8.6 total population-averaged generations). Following selection cells were stored in 1mL of M9 media and 7% (v/v) DMSO at -80°C until bacterial plasmid DNA was extracted using a Qiagen miniprep kit (Qiagen, Valencia, CA).

Kowalsky C.A., Klesmith J.R., Stapleton J.A., Kelly V., Reichkitzer N, & Whitehead T.A. (2015). High-Resolution Sequence-Function Mapping of Full-Length Proteins. PLoS ONE, 10(3), e0118193.

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Determining Bacterial Cell Density and Biomass

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The optical density (OD) was determined at 600 nm using a Genesys 20 spectrophotometer (Thermo Scientific, USA). The value measured from filtered samples was subtracted to compensate for the background of the medium. The cell dry weight (CDW) was determined by filtering appropriate volumes (containing a minimum of 10 mg_CDW and a maximum of 40 mg_CDW on the filter) of cell culture through a pre-dried and weighed 0.45 µm polyethersulfone membrane (Sartorius, Germany). The filters containing samples were washed with deionized water and dried again in a microwave oven at a power output of 385 W for 15 min, before final weighing.

van Dijk M., Mierke F., Nygård Y, & Olsson L. (2020). Nutrient-supplemented propagation of Saccharomyces cerevisiae improves its lignocellulose fermentation ability. AMB Express, 10, 157.

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