Fungal spores were inoculated at 106 spores mL−1 into 40 mL of a primary medium, i.e., glucose minimal medium, in 125-mL flasks containing (per liter) 10 g sucrose, 5 g yeast extract (Difco Laboratories, MD, USA), 50 mL nitrate salts, and 1 mL trace elements, at pH 4.5. The cultures were incubated at 25 °C with shaking at 150 rpm for 48 h. Cultures were harvested by vacuum filtration through a sterile Büchner funnel fitted with a Whatman number 1 filter paper, and the remaining mycelia were washed twice with 50 mL of sterile distilled water. The washed mycelia were resuspended in 50 mL of 0.2 M phthalate-buffered or phosphate-buffered liquid secondary medium (SM) at pH 4 or pH 7, respectively. This SM contained (per liter) 60 g sucrose, 7 g NaNO3, 3 g tryptone (Difco Laboratories, MD, USA), 1 g KH2PO4, 0.5 g MgSO4 · 7H2O, and 0.5 g KCl. The cultures were incubated at 25 °C on a rotating shaker at 150 rpm for 0.5, 1, 3, 10, or 24 h. A sample of mycelia from the cultures was collected into a 1.5-mL Eppendorf at each time point, and the mycelia were frozen with liquid nitrogen for RNA extraction.
Sucrose
Manufactured by BD
Sourced in United States, United Kingdom
Sucrose is a disaccharide sugar composed of glucose and fructose. It is a common ingredient in many laboratory applications involving carbohydrate analysis and biochemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
Agar, by BD (3 mentions)
B chner funnel, by Cytiva (1 mentions)
Whatman filter paper number 1, by Cytiva (1 mentions)
Yeast extract, by BD (1 mentions)
Tryptone, by BD (1 mentions)
2 n morpholino ethanesulfonic acid mes, by Merck Group (1 mentions)
Gamborg s b5 vitamins, by Duchefa Biochemie (1 mentions)
Sucrose, by Merck Group (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Goat serum, by Merck Group (1 mentions)
Triton x, by Merck Group (1 mentions)
Na2hpo4 12h2o, by Merck Group (1 mentions)
Nah2po4 2h2o, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Mgso4 7h2o, by Avantor (1 mentions)
Acetic acid, by BD (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Digoxigenin labeled, by Roche (1 mentions)
Bacto agar, by BD (1 mentions)
Congo red, by Merck Group (1 mentions)
11 protocols using sucrose
1
Fungal Transcriptional Response to pH
2
Germination of P. turczaninovii Achenes
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The germination in the seed boxes of P. turczaninovii seeding material was preliminarily evaluated. Then fruits were surface sterilized by immersion in 70% ethanol for 2 min, followed by immersion for 4 min. in 0.1% (w/v) mercuric chloride (HgCl 2 ) solution containing 3 drops of Tween-80, and then thoroughly rinsed four times with autoclaved distilled water. Aseptic achenes were placed on medium, described as step zero S0, containing ¼ concentration of MS salts (Murashige and Skoog 1962) (link) deprived of phytohormones but supplemented with 0.1 g L -1 myo-inositol, 1 3 0.5 mg L -1 tiamine, 0.5 mg L -1 pyridoxine, 0.5 mg L -1 nicotinic acid, as well as 20 g L -1 sucrose, 0.6 g L -1 activated charcoal, and 5.0 g L -1 agar (Difco Laboratories Inc. UK). The germination percent in vitro on 1/4 MS medium was also checked. Achenes with gentle incised pericarp were placed in 100 mL Erlenmeyer flasks filled with 10 mL of medium. The single experiment consisted of 10 flasks with 10 achenes per flasks, and was repeated twice. Germination was carried out in a growth chamber illuminated with white light during the day, using of 16/8 photoperiod and a temperature of 22/14 °C (day/night). After four weeks the number of aseptic seedlings obtained under in vitro conditions was assessed.
3
Arabidopsis thaliana Floral Dip Transformation
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All Arabidopsis thaliana plants were Columbia (Col-0) ecotype and used for floral dip transformation mediated by Agrobacterium tumefaciens strain GV3101 [53 (link)]. Six growing pots, each containing five Arabidopsis plants, were used for each construct. Seeds were initially stratified for two days at 4 °C, surface sterilized for two min in 75% (v/v) ethanol, and 4 min in 25% (v/v) bleach, and washed with sterile distilled water. Afterwards, they were transferred to soil (Postground P, Klasmann-Deilmann, Geeste, Germany) or plated on Petri dishes containing solid half-strength MS medium (Duchefa Biochemie, Haarlem, The Netherlands) [54 (link)] supplemented with 0.05% MES (2-(N-morpholino) ethanesulfonic acid) (Sigma-Aldrich, St. Louis, MO, USA) pH 5.7, Gamborg’s B5 vitamins, a micronutrient mixture (Duchefa Biochemie, Haarlem, The Netherlands), 2% (w/v) sucrose and 1.2% (w/v) agar (Difco Laboratories, Detroit, MI, USA). The seeds were germinated and grown in a plant growth chamber under long-day conditions at 22 °C (16 h photoperiod).
4
Construction of PAO1 Mutant Strains
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PAO1 mutants were constructed following a previously described protocol (Hmelo et al., 2015 (link)). Briefly, the upstream and downstream regions of each gene open reading frame (ORF) were amplified from PAO1 genomic DNA, digested with KpnI/EcoRI, and ligated into the linearized suicide vector pEX18Gm. The resulting mixtures were transformed into the competent E. coli DH5α cells by electroporation. The constructed recombinant plasmids pEX18Gm-recA, pEX18Gm-lys, pEX18Gm-prtN, pEX18Gm-PA0634, pEX18Gm-PA3866, and pEX18Gm-PA0985 were mobilized from E. coli S17.1 into PAO1 by mating. The single colonies were streaked onto non-salt LB (NSLB) plus 15% sucrose (Sigma-Aldrich) plates and incubated at 30°C for sucrose counterselection. Finally, the sucrose-resistant colonies that grew on LB agar and Pseudomonas Isolation Agar (PIA) plates (BD, Germany) but not on LB agar plates containing 30 μg/ml gentamicin were selected, and the desired mutation was confirmed by PCR and sequencing (Tsingke, Beijing, China). All primers used in this study are listed in Supplementary Table S2 .
5
Immunohistochemical Analysis of Spinal Cord and DRG
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For immunohistochemical studies, spinal columns were dissected and the lumbar part of the spinal cord and DRG from the same level were collected. Neuronal tissue and skin samples were cryoprotected by placing them into a 15% sucrose solution in 0.1 M phosphate buffer (15% wt/vol sucrose [BDH, London, United Kingdom], 3.12 g/L NaH2PO4·2H2O [Sigma, Welwyn Garden City, United Kingdom], and 28.65 g/L Na2HPO4·12H2O [Sigma]) for 24 hours at 4°C. Samples were then transferred to a 30% sucrose solution in 0.1 M phosphate buffer for a further 24 hours before tissue was embedded in optimal cutting temperature compound. Fourteen micrometer (skin and spinal cord) and 7 μm (DRG) sections were sectioned on a cryostat and thaw mounted onto glass slides.
For immunohistochemistry, slide-mounted tissue sections were blocked with 10% goat serum (Millipore, Hertfordshire, United Kingdom) in PBS containing 0.2% Triton-X (Sigma) and 0.1% sodium azide (Sigma) (PBS-X). Slides were then incubated overnight at room temperature with primary antibody in PBS-X followed by the appropriate secondary antibody for 2 hours (Table2 ). At the end of the protocol, slides were coverslipped with mounting medium (VectaShield, Peterborough, United Kingdom).
For immunohistochemistry, slide-mounted tissue sections were blocked with 10% goat serum (Millipore, Hertfordshire, United Kingdom) in PBS containing 0.2% Triton-X (Sigma) and 0.1% sodium azide (Sigma) (PBS-X). Slides were then incubated overnight at room temperature with primary antibody in PBS-X followed by the appropriate secondary antibody for 2 hours (Table
6
Inoculum Preparation for EPS Production
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For inoculum preparation, S. kilbournensis was inoculated in SmF in Luria broth medium supplemented with (g/L) sucrose (BD Bioxon®, Mexico City, Mexico) (6), peptone (Hycel®, Mexico City, Mexico) (1), (NH4)2SO4 (Meyer®, Mexico City, Mexico) (0.2), KH2PO4 (Fermont™, Mexico City, Mexico) (0.1), and MgSO4·7H2O (J.T. Baker®, Mexico) (0.1) with an adjusted initial pH of 6.8 and was maintained at 30 °C at 150 rpm for 24 h. For biomass recuperation, the culture was centrifuged at 3500× g for 15 min; then, the pellet was resuspended in distilled water, and the number of cells was determined using a Neubauer chamber. The suspension obtained was considered the SmF inoculum for the production of EPSs and was stored at 4 °C until use.
7
Ethanol Production from Sucrose and Yeast
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Sucrose was obtained from BDH, acetic acid from El Naser Company, Sodium acetate and all the other chemicals used were obtained from Merck. The commercial live bakers' yeast, Saccharomyces cerevisiae was obtained, in two forms, one form of active dry yeast, from the Egyptian company for advanced Foodstuff Industries. The fresh compressed yeast brought from Sugar Company of integrated industries, Egypt. Baker's yeast used without further purification .Sawdust samples were obtained from local sawmills.
8
Fluorescent Labeling and Histological Analysis of Zebrafish Embryos
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Probes were synthesized with either digoxigenin-labeled or fluorescine-labeled RNA antisense oligonucleotides (Roche, USA). WISH was performed according to published methods49 . Anti-phosphohistone 3 antibodies (1:500, Millipore, USA) and anti-mouse Alexa fluor 488 stained for proliferating cells in 24 hpf whole-mount embryos. Cell death in 24 hpf and 48 hpf embryos were detected with TUNEL assay TMR red (Roche, USA). For cryosectioning, 4% PFA fixed embryos were embedded and oriented in 2% bactoagar (BDH, USA) and soaked in 30% sucrose (BD, USA) overnight. The agar blocks were trimmed and covered with Tissue-Tek O.C.T medium (Sakura, Japan) before being sectioned at 15 μm thickness using Microm HM 505 cryostat chamber (Carl Zeiss, Germany). The sections were collected with PolysineTM coated slides (Menzel GmbH and Co, Germany), and labelled for proliferating cells with anti-phosphohistone 3 antibodies and anti-mouse POD at a dilution of 1:500, before being stained with DAB (2,4-Diaminobutyric Acid) (Sigma-Aldrich, USA).
9
MRSA Colony Morphologies on CRA
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Colony morphologies and phenotypic changes researched utilizing CRA, as recently portrayed. The CRA was made out of 37 g/L of cerebrum heart implantation stock (Himedia), 36 g/L of sucrose,15 g/L of agar (BD Biosciences, Franklin Lakes, NJ, USA), and 0.8 g/L of Congo red (Sigma, St. Louis, MO, USA). MRSA cells on CRA were brooded with and without HAP-Se-HSPD for 24 h at 37°C before taking pictures.
10
In Vitro Cultivation and Stress Treatments of Vitis amurensis
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Vitis amurensis plantlets were grown under in vitro conditions on solid medium (half-strength B5 (Duchefa Biochemie, Haarlem, Niederlande) solid medium supplemented with 0.2 mg l−1 IBA (Phyto Tech, Lenexa, Kansas, USA), 30 g l−1 sucrose (Beijing Chemical Works, Beijing, China) 0.6% agar (BD, Franklin Lakes, NJ, USA), pH 5.8) and liquid medium (half-strength B5 solid medium supplemented with 0.2 mg l−1 IBA, 30 g l−1 sucrose, pH 5.8) in culture flasks (300 ml) at 25°C under a 16-h light/8-h dark photoperiod and 100 μmol m−2 s−1 light intensity. Six-week-old V . amurensis plantlets were then exposed to solid medium at 4°C for cold stress treatment and liquid medium supplemented with 8% PEG-6000 (BioRular, Danbury, CT, USA) for osmotic stress treatment. The same plantlets with no treatments were used as control. There were three biological replicates in different treatments.
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Vitis amurensis plantlets were grown under in vitro conditions on solid medium (half-strength B5 (Duchefa Biochemie, Haarlem, Niederlande) solid medium supplemented with 0.2 mg l−1 IBA (Phyto Tech, Lenexa, Kansas, USA), 30 g l−1 sucrose (Beijing Chemical Works, Beijing, China) 0.6% agar (BD, Franklin Lakes, NJ, USA), pH 5.8) and liquid medium (half-strength B5 solid medium supplemented with 0.2 mg l−1 IBA, 30 g l−1 sucrose, pH 5.8) in culture flasks (300 ml) at 25°C under a 16-h light/8-h dark photoperiod and 100 μmol m−2 s−1 light intensity. Six-week-old V . amurensis plantlets were then exposed to solid medium at 4°C for cold stress treatment and liquid medium supplemented with 8% PEG-6000 (BioRular, Danbury, CT, USA) for osmotic stress treatment. The same plantlets with no treatments were used as control. There were three biological replicates in different treatments.
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