Spectrophotometric analysis of pigments was performed by an Ultrospec 2100 Pro spectrophotometer (GE Healthcare Ltd., Little Chalfont, England) following the method described by Porra et al. [28 (link)], with minor modifications. Fresh samples (0.3 g) were extracted in 20 mL of acetone 80% and agitated in the dark at 4 °C for 3 days. The chlorophyll and carotenoid content were determined by the increase in absorbance at 663 nm for chlorophyll a, 648 nm for chlorophyll b and 470 nm for carotenoids against a blank solution of acetone 80%. Total chlorophylls and carotenoids were expressed as mg g−1 fresh weight (FW).
Ultrospec 2100 pro spectrophotometer
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Japan, Sweden
The Ultrospec 2100 pro spectrophotometer is a compact and versatile laboratory instrument designed for accurate absorbance measurements across a wide range of applications. It features a wavelength range of 190 to 900 nm and can perform single-wavelength, multi-wavelength, and scanning measurements.
Automatically generated - may contain errors
14 protocols using ultrospec 2100 pro spectrophotometer
1
Spectrophotometric Analysis of Plant Pigments
2
Biochemical Analysis of Biomass Composition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipid, protein, and carbohydrate were analyzed using traditional biochemical methods to validate the FT-IR results. Lipid content analysis was performed according to our previous method [7 (link)]. The lyophilized cell was ground by mortar and pestle into a fine powder under liquid nitrogen. The cell powder was extracted into 100 mL of chloroform/methanol (2:1, v/v) at room temperature. The lipid extract was dried by evaporation and weighed. Carbohydrate content was analyzed using sulfuric acid-anthrone method [16 (link)]. Absorbance was measured at 620 nm with Ultrospec 2100 pro Spectrophotometer (GE, Boston, MA, USA). A calibration curve was prepared using glucose as a standard. Protein content was analyzed using the Bradford method [15 (link)]. Fatty acids analysis was performed based on our previous method [7 (link)].
3
Bacterial Strains and Plasmids for Aminoglycoside Resistance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bacterial strains and plasmids used in this study are listed in Table 1 . Of note various P. aeruginosa strains in which contribution of the MexXY efflux system to aminoglycoside resistance were assessed before (Morita et al., 2012a (link)) are used in the main (PAO1; a reference strain, PAGU 1498 and PAGU 1606; multidrug resistant clinical isolates, PAGU 1569; pan-aminoglycoside clinical isolates) (Table 1 ). Among the four strains PAGU 1498 is the agrZ-type MexXY-overproducing mutant (Morita et al., 2012a (link)).
Bacterial cells were grown (unless otherwise indicated) in Luria (L) broth and on L agar (1.5%) under aerobic conditions at 37°C, as previously described, with antibiotics as specified (Morita et al., 2015b ). Bacterial growth was quantified by measuring the optical density at 600 nm on an Ultrospec 2100 Pro Spectrophotometer (GE Healthcare Corp., Tokyo, Japan), unless otherwise indicated. Cells harboring the plasmid pEX18Tc (Hoang et al., 1998 (link)) or derivatives thereof were maintained on medium supplemented with 2.5–10 μg/ml tetracycline for E. coli or and selected on medium supplemented with 20–150 μg/ml tetracycline for P. aeruginosa. Cells harboring the plasmid pFLP2 (Hoang et al., 1998 (link)) were maintained and selected on medium supplemented with 100 μg/ml ampicillin for E. coli or 50–200 μg/ml carbenicillin for P. aeruginosa.
Bacterial cells were grown (unless otherwise indicated) in Luria (L) broth and on L agar (1.5%) under aerobic conditions at 37°C, as previously described, with antibiotics as specified (Morita et al., 2015b ). Bacterial growth was quantified by measuring the optical density at 600 nm on an Ultrospec 2100 Pro Spectrophotometer (GE Healthcare Corp., Tokyo, Japan), unless otherwise indicated. Cells harboring the plasmid pEX18Tc (Hoang et al., 1998 (link)) or derivatives thereof were maintained on medium supplemented with 2.5–10 μg/ml tetracycline for E. coli or and selected on medium supplemented with 20–150 μg/ml tetracycline for P. aeruginosa. Cells harboring the plasmid pFLP2 (Hoang et al., 1998 (link)) were maintained and selected on medium supplemented with 100 μg/ml ampicillin for E. coli or 50–200 μg/ml carbenicillin for P. aeruginosa.
4
Nicotine Degradation Enzyme Kinetic Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All assays were carried out in quartz cuvettes (1-cm light path) filled with 1 mL of reaction mixture at 30 °C using a UV–visible Ultrospec 2100 pro Spectrophotometer (GE Healthcare, USA). The reactions were initiated by the addition of enzyme. Ndh activity was determined as previously described [11 (link)] by monitoring the reduction of 2,6-dichlorophenolindophenolsodium (DCIP) with nicotine at 600 nm (ε = 21 mM−1 cm−1). The assay mixture contained 1 mM nicotine, 0.05 mM DCIP, and 50 mM phosphate buffer (pH 7.0). Hno activity was measured by detecting the formation of 6-hydroxypseudooxynicotine as previously reported [36 (link)]. The assay mixture contained 0.56 mM 6-hydroxynicotine, 100 mM NaCl, and 100 mM glycine–NaOH buffer (pH 9.2). The formation of 6-hydroxypseudooxynicotine was followed at 334 nm (ε = 20.7 mM−1 cm−1). One unit (U) of enzyme activity was defined as the amount of enzyme catalyzing the conversion 1 µmol of substrate per minute. Protein concentration was measured using the Bradford assay with bovine serum albumin as the standard [37 (link)].
5
Bacterial Growth Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial strains and plasmids used in this study are listed in Table 1 . Pre-cultures of E. coli and C. glutamicum were carried out in 2xTY medium in baffled Erlenmeyer flasks on a rotary shaker (130 rpm) at 30 and 37°C, respectively. CgXII was used as minimal medium for C. glutamicum (Eggeling and Bott, 2005 (link)) with 10 g L-1 glucose as carbon source. Strains carrying plasmids were cultivated in the presence of kanamycin (50 μg/mL) and IPTG (1 mM) for mBFP expression. Growth of E. coli and of C. glutamicum was followed by measuring the optical density (OD) at 600 nm in an Ultrospec 2100 pro spectrophotometer (GE Healthcare Life Sciences GmbH, Freiburg, Germany).
6
Colorimetric Assay of α-L-Fucosidase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The activity of α-l -fucosidase was measured using a colorimetric assay with 2-chloro-nitrophenyl α-l -fucopyranoside (CNPF; Carbosynth Limited, Compton, UK) as a substrate. Spent culture medium (10 μl) was added to 100 μl of a 20-mM potassium acetate solution (pH 5.5) containing 2 mM CNPF. This mixture was incubated at 37°C for ∼15 h. The reaction was stopped by the addition of 50 μl of 1 M sodium carbonate, and the absorbance of released 2-chloro-4-nitrophenol was measured at 405 nm using an Ultrospec 2100 pro spectrophotometer (GE Healthcare).
7
Growth Monitoring of M. xanthus Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
M. xanthus cells were grown to exponential phase, diluted with fresh medium to an OD550 of 0.025, and transferred in 24-well polystyrene microtiter plates. Growth was then monitored in an Infinite® M1000 PRO scanner (Tecan) by measuring the optical density at 550 nm (OD550) at 15 min intervals, with three replicates per strain. Alternatively, cells were grown in Erlenmeyer flasks, sampled manually at defined intervals, and analyzed in an Ultrospec 2100 pro spectrophotometer (GE Healthcare).
+ Expand
M. xanthus cells were grown to exponential phase, diluted with fresh medium to an OD550 of 0.025, and transferred in 24-well polystyrene microtiter plates. Growth was then monitored in an Infinite® M1000 PRO scanner (Tecan) by measuring the optical density at 550 nm (OD550) at 15 min intervals, with three replicates per strain. Alternatively, cells were grown in Erlenmeyer flasks, sampled manually at defined intervals, and analyzed in an Ultrospec 2100 pro spectrophotometer (GE Healthcare).
8
Bacterial Culture and Plasmid Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bacterial strains and plasmids used in this study are listed in Table 1 . Bacterial cells were grown in Luria (L) broth and on L agar (1.5%) under aerobic conditions at 37°C as previously described, unless otherwise indicated, with antibiotics as necessary (Morita et al., 2010 (link)). Bacterial growth was quantified by measuring the optical density at 600 nm on an Ultrospec 2100 Pro Spectrophotometer (GE Healthcare Corp., Tokyo, Japan), unless otherwise indicated. The plasmids pEX18Tc (Hoang et al., 1998 (link)), pYM101 (Morita et al., 2010 (link)), and their derivatives were maintained and selected using medium supplemented with 2.5-10 μg tetracycline ml−1 for E. coli or 50–150 μg tetracycline ml−1 for P. aeruginosa. The plasmids pUCP20T (Schweizer et al., 1996 ) and pFLP2 (Hoang et al., 1998 (link)) and their derivatives were maintained and selected using medium supplemented with 100 μg ampicillin ml−1 for E. coli or 200 μg carbenicillin ml−1 for P. aeruginosa.
9
SADH Activity Measurement in Cell Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extract was prepared with Yeast Protein Extraction Reagent (Thermo Scientific, Pierce, Rockford, USA) according to the instructions provided by the manufacturer. The total protein concentration in cell extracts was determined using the Bradford method [34 (link)] with bovine serum albumin (BSA) as standard. SADH activity measurements were performed as described previously [35 (link)]. The activity is based on measuring the oxidation of NADH at 340 nm with an Ultrospec 2100 pro spectrophotometer (GE Healthcare Life Sciences, Sweden). The data were collected with the software program SWIFTII (Amersham Biosciences, Sweden). Cell extracts were diluted until the decrease in absorbance was linear for 5 min, at which point the activity could be calculated from the slope. One unit of activity corresponds to 1 µmol NADH consumed per minute at 25 °C. The assay contained sodium phosphate buffer (50 mM, pH 7), acetophenone (10 mM), NADH (0.2 mM), and cell extract (1-30 mg/l total protein).
10
Expression Analysis of Terpenoid Biosynthesis Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from each sample by the guanidine thiocyanate procedure [22 (link)]. The concentration and quality of RNA were measured using agarose gel electrophoresis and an Ultrospec 2100 pro spectrophotometer (GE, Fairfield, CT, USA). The first cDNA strand was synthesized using the GoTaqTM Reverse Transcription System (Promega, Madison, WI, USA). The specific primers were designed by Primer Premier 5.0, as previously described [23 (link)]. 18S rRNA served as the reference gene. The quantitative reaction was conducted using GoTaq® qPCR Master Mix Real-Time PCR System (Promega, USA) for 40 cycles with the following reaction conditions: 2 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s (96, Roche, Swiss). The expression levels of PnCAS (cycloartenol synthase), PnDS (dammarenediol synthase), PnHMGR (3-hydroxy-3-methyl-glutaryl coenzyme A reductase), PnFPS (farnesyl pyrophosphate), PnSS (squalene synthase), PnSE (squalene epoxidase), and PnbHLH1 were analyzed (Table 1 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!