For each tree, insect DNA extraction was performed on two randomly selected EAB adults. The wings were first removed with sterile tweezers and scissors, and the exoskeleton was sterilized by agitating (Fisher Vortex Genie 2, Ottawa, ON, Canada) the beetle in 1 mL of 70% ethanol for 1 min. Beetles were rinsed with 1 mL of sterile water by vortexing for 30 s. Dissection was performed in sterile phosphate-buffered saline, the two guts were pooled, and total genomic DNA was extracted by the mechanical lysis as described by Mogouong, et al.11 (link). For each leaf sample, endophytes and epiphytes were processed together by grinding five leaflets corresponding to the apical leaflet of five leaves randomly selected (Supp. Fig. S4 ) in liquid nitrogen and using the MoBio PowerSoil DNA isolation kit (Qiagen, Toronto, ON, Canada). DNA extracts from insect guts and leaves were purified using a PowerClean Pro DNA clean-up kit (Qiagen, Venlo, the Netherlands). DNA concentration was estimated using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Life Technologies, Burlington, ON, Canada) following the manufacturer’s instructions.
Vortex genie 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vortex Genie 2 is a laboratory vortex mixer designed to mix and agitate samples. It features variable speed control and can accommodate a variety of sample containers.
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Lab products found in correlation
Powerclean pro dna clean up kit, by Qiagen (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Sigma 3 18 k, by Merck Group (1 mentions)
Qtof 6530, by Agilent Technologies (1 mentions)
Buffered peptone water broth, by 3M (1 mentions)
Porcine skin gelatin, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Freeze dryer, by Labconco (1 mentions)
Hplc gradient grade acetonitrile, by Merck Group (1 mentions)
Polypropylene centrifuge tubes, by Avantor (1 mentions)
L phenylalanine, by Merck Group (1 mentions)
13 protocols using vortex genie 2
1
Insect DNA Extraction from Emerald Ash Borer
2
Prednisolone Saturation Protocol
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The prednisolone was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the absolute ethanol was purchased from Scharlau (Barcelona, Spain). The saturated prednisolone solution was prepared by putting the excess amount of prednisolone into absolute ethanol. The addition of prednisolone was stopped only if oversaturation was observed after shaking of 10 mins (Fisher Vortex Genie 2, Hampton, NH, USA). The oversaturated solution was then centrifuged at 14,000 rpm for 5 min (Sigma 3-18K, Tokyo, Japan) and the saturated prednisolone was removed and ready for use. The prednisolone solution was later used to mix with deionized (DI) water in the microchannel.
3
Quantitative Bile Acid Analysis by LC-MS
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Internal standard (13C6-DCF) was added to 25 µl coculture medium to give a final concentration of 20 μM DCF when at 50–100× Cmax (4.4 µM was Cmax for our study), and 2 μM with DCF at 1× Cmax of d5-GCA (0.5–1 μM) was added as an internal standard prior to sample extraction for bile acid measurements. The choice was arbitrary. 4.4 is a low value in the range of the physiologic dose, and higher values were chosen to represent overdosing. MeOH was then added at a 1:4 ratio [(v/v); 25 µl sample/100 µl MeOH). Resulting suspensions were maintained at −20°C for 5 minutes, vortexed for 20 seconds, and subjected to gentle shaking for 5 minutes on a Fisher Vortex Genie 2 with a vortex adapter. The samples were then maintained at −20°C for 5 minutes and centrifuged at 15,000 rpm for 10 minutes. The supernatants were then collected carefully (without disturbing the protein pellet) and dried in a SpeedVac (Savant Instruments, Holbrook, NY). Samples were prepared immediately for liquid chromatography (LC)–mass spectrometry (MS) analysis by resuspension in 2% ACN containing 0.1% FA. Injections of 1–5 µl were analyzed on an Agilent QTOF 6530 using parameters described previously (Sarkar et al., 2015 (link)).
4
Silica Encapsulation of Phosphorescent Particles
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Typically, 1.6 mg of dry milled
size-fractionated phosphorescent particles was suspended in 1 mL of
anhydrous ethanol in a 2 mL microcentrifuge tube. A solution containing
approximately 222 μL of ethanol, 247 μL of DI water, and
6.7 μL of TEOS was prepared, vigorously agitated, and then added
immediately to the suspension of phosphors in ethanol. The tube with
the particles was then placed in a bath sonicator (Fisher Scientific
FS30) for 5 min, and afterward, 25 μL of aqueous ammonium hydroxide
(28–30%) was added to the reaction mixture, bringing the final
volume to 1.5 mL. The calculated concentrations of TEOS and ammonia
were 20 mM and 0.25 M, respectively, with approximately 17.5% v/v
water and 81.4% v/v ethanol. After adding the ammonium hydroxide,
the particles were sonicated for an additional 30 min and then placed
on a room temperature rotator for 7.5 h to keep the particles suspended,
with an overall silica encapsulation time of approximately 8 h. Silica
encapsulation was stopped by centrifuging to induce settling (Eppendorf
Centrifuge 5418), removing the supernatant, and resuspending the particles
in ethanol by vigorous agitation with a Fisher Vortex Genie 2 and
sonication. After 4 wash cycles, the particles were dried in a Savant
SpeedVac Concentrator SVC100H at 36 °C under reduced pressure
for 1 h.
size-fractionated phosphorescent particles was suspended in 1 mL of
anhydrous ethanol in a 2 mL microcentrifuge tube. A solution containing
approximately 222 μL of ethanol, 247 μL of DI water, and
6.7 μL of TEOS was prepared, vigorously agitated, and then added
immediately to the suspension of phosphors in ethanol. The tube with
the particles was then placed in a bath sonicator (Fisher Scientific
FS30) for 5 min, and afterward, 25 μL of aqueous ammonium hydroxide
(28–30%) was added to the reaction mixture, bringing the final
volume to 1.5 mL. The calculated concentrations of TEOS and ammonia
were 20 mM and 0.25 M, respectively, with approximately 17.5% v/v
water and 81.4% v/v ethanol. After adding the ammonium hydroxide,
the particles were sonicated for an additional 30 min and then placed
on a room temperature rotator for 7.5 h to keep the particles suspended,
with an overall silica encapsulation time of approximately 8 h. Silica
encapsulation was stopped by centrifuging to induce settling (Eppendorf
Centrifuge 5418), removing the supernatant, and resuspending the particles
in ethanol by vigorous agitation with a Fisher Vortex Genie 2 and
sonication. After 4 wash cycles, the particles were dried in a Savant
SpeedVac Concentrator SVC100H at 36 °C under reduced pressure
for 1 h.
5
Rapid Aerobic Plate Counting of Steak
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Total plate count was determined on dark-cutting steak in PVC and nitrite-embedded treatment on day 3 of display. A sterile 5 × 5 cm2 grid was utilized to swab the surface of each steak with a 3M Swab-Sampler with 10 mL buffered peptone water broth (3M Maplewood, MN). Swab containers were vortexed for 30 s utilizing a Fisher Scientific Vortex-Genie 2 (12–812; Hampton, NH). One mL of the swabbed sample was serially diluted in 9 mL of 0.1% sterile peptone water and one mL of each dilution was aseptically plated on 3M Petrifilm rapid aerobic count plates (Hampton, NH). Plates were incubated in a VWR Forced Air General Incubator (5.4 ft3; VWR, Radnor, PA) at 37°C for 48 h. Following incubation, plates were counted on an Interscience Scan 100 pressure sensitive pad (Interscience, Woburn, MA) to determine total plate count per cm2.
6
Yeast RNA Extraction and qPCR Analysis
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2 ml yeast cultures were grown to midlog (OD600 ∼ 0.3–0.6) after which RNA was extracted using the Trizol RNA extraction method. Briefly, cells were centrifuged at 4000 rpm for 15 min followed by resuspension in 1 ml of Trizol reagent. Glass beads were added to the microfuge tubes and cells were disrupted using the Fisher Vortex Genie 2 for 5 min at 4°C. 200 μl chloroform was added, vortexed for 15 s followed by a 5 min incubation at RT. Tubes were centrifuged at 13 300 rpm for 5 min at 4°C. Aqueous layer was recovered followed by another chloroform extraction. RNA was precipitated by adding 500μl of isopropanol and incubating on ice for 15 min. Tubes were centrifuged at 13 300 rpm for 15 min at 4°C. Pellets were washed with 70% ethanol and re-dissolved in 50μl RNase free distilled water. qPCR was carried out using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit as per manufacturer's instructions using oligos mentioned in Supplemental Table S1. Control experiments were carried out to assess the specificity of the probes used.
7
Wastewater Sample Preparation for LC-MS/MS
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Triplicate wastewater samples collected in 50 mL polypropylene centrifuge tubes were vortexed (Vortex Genie 2, Fisher, NY, USA) for 10 s before being transferred to smaller tubes. Then, 1.8 mL of the wastewater was transferred to 2 mL microcentrifuge tubes and centrifuged (Eppendorf 5415D, Hamburg, Germany ) at 12,000 rpm for 15 min. After centrifugation, 1.5 mL of the wastewater supernatant and 1.5 mL MeOH were mixed in 5 mL glass tubes. The mixture was vortexed for 10 s and 1.5 mL was filtered through 0.2 µm syringe filter (Acrodisc with PTFE membrane, Waters, MA, USA). Extracts were stored at -20°C until analysis with the high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
8
Chemical Lysis for Bacterial DNA Extraction from Sputum
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Chemical lysis was performed to release the bacterial DNA into the sputum [20 –22 (link)]. In 2 mL tubes, 500 uL of surrogate sputum were mixed with 500 uL of lysis and binding buffer (4M guanidine thiocyanate, 25 mM sodium citrate, 4.9% Triton X-100, 0.2% sodium dodecyl sulfate) and 0.8 mg of MyOne Silane Dynal beads. This mixture was agitated for 10 minutes on a Fisher Vortex Genie 2 at speed 4. After agitation, 1 mL of lysed sample was pipetted into the tubing for extraction (Fig 1 ). The procedure was repeated for each concentration of TB in surrogate sputum: 0, 103, 104, and 105 cells/mL.
9
Bacterial Cell Surface Adhesion Assay
10
Fabrication of PEGDA and GelMA Hydrogels
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