Strain CF-236885 (F. fallens) was fermented by inoculating ten mycelial agar plugs into SMYA medium (Bacto neopeptone 10 g; maltose 40 g; yeast extract 10 g; agar 3 g; H2O 1 L) in a flask (50 mL medium in a 250 mL Erlenmeyer). The flask was incubated on a rotary shaker at 220 rpm at 22 °C with 80% relative humidity. After growing the seed stage for seven days, a 1.5 mL aliquot was used to inoculate each flask of the production medium CMK (D-cellobiose 40 g; yeast extract 1 g; glycerol 2 g; Murashige and Skoog salts (SIGMA M-5524) 4.3 g, and distilled water 1 L). The 20 flasks (50 mL medium per 250 mL unbaffled flask) were incubated at 22 °C on a rotary shaker (220 rpm, 5 cm throw) for 23 days, 80% relative humidity.
M5524
Manufactured by Merck Group
Sourced in United States, Germany
The M5524 is a versatile laboratory equipment designed for a range of scientific applications. It is a compact and durable device that can be used for various laboratory tasks. The core function of the M5524 is to provide a reliable and consistent performance in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
A1296, by Merck Group (1 mentions)
Luck 100, by GoldBio (1 mentions)
Ms solid medium, by Merck Group (1 mentions)
Kinetin, by Merck Group (1 mentions)
1 naphthaleneacetic acid, by Merck Group (1 mentions)
2 4 dichlorophenoxyacetic acid, by Merck Group (1 mentions)
Growth chamber, by Percival (1 mentions)
Growth chamber, by Conviron (1 mentions)
Metro mix 360, by Sun Gro Horticulture (1 mentions)
M8250, by Merck Group (1 mentions)
Plant agar, by Merck Group (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
21 protocols using m5524
1
Fermentation and Production of Fungal Metabolites
2
Arabidopsis Transformation by Floral Dip
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3
Arabidopsis Transformation by Floral Dip
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4
Arabidopsis Transformation with FveSEP3 Variants
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Arabidopsis Col-0 was transformed with FveSEP3-ox and FveSEP3G27E-ox in Agrobacterium tumefaciens GV3101 using the floral-dip method. The T1 transgenic lines were selected on half-strength MS (M5524, Sigma-Aldrich) with 100 mg l−1 kanamycin.
5
Arabidopsis Salinity Stress Assay
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Arabidopsis thaliana Col-0 (wild-type) seeds were surface sterilized by shaking for 10 min in 70% ethanol + 0.05% sodium dodecyl sulfate (SDS), then washed twice with 99% ethanol and once with sterilized H2O. The seeds were then sown on square Petri dishes (12x12 cm) containing half-strength Murashige and Skoog Basal Salt Mixture pH 5.8, 0.9% agar (½MS) [45 ] (M5524, Sigma Aldrich, Germany) without sucrose. The plates were stored in the dark for 2 days at 4°C for seed stratification and then incubated vertically (~75° angle to the horizontal) in growth chambers (Percival Scientific Inc., USA) at 22°C with a photoperiod of 16/8 h (light/dark) for germination. 5-day old seedlings (~1–1.5 cm in root lengths) were then gently transferred to fresh ½MS agar plates supplemented with 100 mM NaCl as a salinity stress (5 seedlings/plate). A “lawn” of bacterial isolates were spread on LB agar plates and incubated at 28°C 24 hours prior to transfer of seedlings. From these plates, square-shaped (3x3 mm) plugs were cut out and laid beside the root system of each seedling without any physical damage (bacteria-free LB agar plugs were used as a mock control) (Panel D in S1 Fig ). For assessment of the effect of inoculation with cultured bacteria, images of representative plants were taken 16 days after transfer (DAT) and compared to mock (bacteria free LB control).
6
Inducing Sporulation in Fungal Isolates
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To induce sporulation, isolates were cultured onto different media in 9-cm Petri dishes; potato-dextrose agar (PDA), synthetic nutrient-poor agar (SNA), malt extract agar (MEA), oatmeal agar (OA) (see Crous et al. 2009 ), MMN (Marx 1969 ) and Murashige-Skoog agar (MS, Murashige & Skoog 1962 , M5524 Sigma-Aldrich Co. LLC., USA) were used. All cultures were incubated at room temperature in the dark, while cultures on MMN and MS were also incubated in the dark at 10 °C.
Furthermore, isolates were also cultured onto the following autoclaved plant parts laid on media to promote sporulation: barley shoots, pine needles, stinging nettle stems and rye grass roots on SNA, and white elm stems on MS. Two parallel replicates of each representative isolate studied were incubated at room temperature and at 10 °C in the dark.
Three parallel replicates of each representative isolate of the groups were cultured onto MEA and MMN and three different treatments applied: i) colonies were burned with a red-hot needle and incubated for 4 wk at room temperature; ii) colonies were exposed daily to near-ultraviolet light for 3 × 10 min for 3 d and subsequently incubated for 4 wk at room temperature, and afterwards stored in dark at 4 °C for several months; and iii) colonies were allowed to dry out on the laboratory bench over a period of 3 mo at room temperature.
Furthermore, isolates were also cultured onto the following autoclaved plant parts laid on media to promote sporulation: barley shoots, pine needles, stinging nettle stems and rye grass roots on SNA, and white elm stems on MS. Two parallel replicates of each representative isolate studied were incubated at room temperature and at 10 °C in the dark.
Three parallel replicates of each representative isolate of the groups were cultured onto MEA and MMN and three different treatments applied: i) colonies were burned with a red-hot needle and incubated for 4 wk at room temperature; ii) colonies were exposed daily to near-ultraviolet light for 3 × 10 min for 3 d and subsequently incubated for 4 wk at room temperature, and afterwards stored in dark at 4 °C for several months; and iii) colonies were allowed to dry out on the laboratory bench over a period of 3 mo at room temperature.
7
Hpa Spore Germination Assay
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MS medium (Murashige and Skoog, 1962 ) was prepared with 4.3 g/L MS basal salt mixture powder (Sigma, M5524), agar (1.5%) (Sigma, A1296), sucrose (10 g/L) (Sigma, 84100), and distilled water. MS powder was dissolved in sterile distilled H2O, the pH was adjusted to 5.7 using 1 M NaOH/HCl, and agar and sugar were added. The medium was sterilized by autoclaving at 15 psi and 121 °C for 15 min. Approximately 20 mL of the medium was aliquoted into each sterile Petri dish in a laminar airflow unit.
Cellophane strips, 1.5 cm in length, were cut from plain transparent florists' cellophane and autoclaved in distilled water. After autoclaving, cellophane strips were placed onto the MS medium in Petri dishes under a laminar airflow and dishes were kept in the fridge for long‐term storage.
Hpa spores were collected, washed twice in sterile distilled water and the spore concentration was adjusted to 5 × 104 spores/mL using a haemocytometer. Approximately 10 µL spore suspension, with 0 or 20 µM antisense sRNA, was dropped onto each piece of cellophane. Plates were incubated with a 12 h light/12 h dark regime at 16 °C. Spores were examined under a light microscope 48 h after incubation and germinated spores were counted.
Cellophane strips, 1.5 cm in length, were cut from plain transparent florists' cellophane and autoclaved in distilled water. After autoclaving, cellophane strips were placed onto the MS medium in Petri dishes under a laminar airflow and dishes were kept in the fridge for long‐term storage.
Hpa spores were collected, washed twice in sterile distilled water and the spore concentration was adjusted to 5 × 104 spores/mL using a haemocytometer. Approximately 10 µL spore suspension, with 0 or 20 µM antisense sRNA, was dropped onto each piece of cellophane. Plates were incubated with a 12 h light/12 h dark regime at 16 °C. Spores were examined under a light microscope 48 h after incubation and germinated spores were counted.
8
Analyzing Circadian Rhythms in Arabidopsis
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Experiments were conducted in Arabidopsis thaliana (Columbia, RRID: SCR_004618). Arabidopsis seeds were surface sterilized and sown on soil or 0.8% agar plates containing half‐strength MS medium (Sigma‐Aldrich M5524). CCR2::LUC and phyB‐9 CCR2::LUC lines have previously been reported (Jones, Hu, Litthauer, Lagarias, & Harmer, 2015 ). A wild‐type Columbia line expressing CCA1::LUC2 (Jones et al., 2015 ) was crossed with either cry1‐304 cry2‐1 (Mockler, Guo, Yang, Duong, & Lin, 1999 ) or phyA‐211 (Reed, Nagatani, Elich, Fagan, & Chory, 1994 ) to obtain cry1‐304 CCA1::LUC2, cry2‐1 CCA1::LUC2, cry1‐304 cry2‐1 CCA1::LUC2, and phyA‐211 CCA1::LUC2 seedlings. Plants were entrained under 12‐hr‐white‐light/12‐hr‐dark cycles under 60 μmol m−2 s−1 before circadian imaging. Red (~600–700 nm, peaking at ~660 nm) and blue (~420–510 nm, peaking at ~450 nm) light was provided by light‐emitting diodes (LEDs; Bright Technology Industrial Ltd., Shenzhen City, China). Green light was provided by LEDs that were filtered through Schott OG515 glass producing a spectral range of ~500–600 nm peaking at ~530 nm (illustrated in Figure 1 a).
9
Bioluminescent Imaging of BY-2 Cells
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Transformations of BY-2 cells were made by agrobacterial strains encoding plasmids. Fifty hours after infiltration, BY-2 cells were supplemented with 150 μl of Murashige and Skoog medium (M5524, Sigma-Aldrich; pH 5.7), containing 100 μM d -Ln (LUCK-100, GoldBio) in the case of FFLuc and Nano-Glo Live Cell substrate (kit N2011, Promega) in case of NanoLuc or no substrate in the case of autoluminescent systems. Plates were imaged in Tecan Spark imager with an open filter and automatic attenuation at 0.1-s exposure times for 60 min. Data processing was performed using custom Python scripts. Integral signal was quantified by integration along the time axis using the composite trapezoidal rule (trapz function from numpy Python package, v1.22.4).
10
Surface Sterilization and Stratification of Arabidopsis Seeds
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Seeds of A. thaliana mutant lines used in our study were ordered from the European Arabidopsis Stock Centre (NASC) or kindly donated by authors (see Supplementary Table S1 for further details). Seeds were surface sterilised using chlorine gas for 4 h, and then stratified for at least 3 days at 4 °C in water. Seeds were sown on square plates (Greiner Bio One; 120 × 120 × 17 mm) containing 75 ml solid ½ strength Murashige-Skoog medium (pH 5.7) (Sigma-Aldrich, M5524 supplemented with 2% sucrose and 1% agar). Plates were sealed and placed in a vertical position into a controlled growth chamber (VB1514 Vötsch Industrietechnik) under a 16 h photoperiod (75 µmol m−2 s−1) at 22 °C/18 °C for 10 days.
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