To produce dsRNA, a single colony of transformed bacteria was cultured in Luria-Bertani (LB) containing 100 μg/ml ampicillin at 37°C with shaking at 250 rpm for 16 h. The culture broth (5 ml) was added to 500 ml of fresh LB medium containing 100 μg/ml ampicillin and allowed to grow at 37°C until it reached an exponential bacterial growth phase with absorbance of 0.6~0.7 at 600 nm. Expression of T7 polymerase was induced by adding 0.4 mM (final concentration) of isopropyl thiogalactose (IPTG) and then further cultured at 30°C for 6 h. Total bacterial RNA was extracted with an RNA Extraction Mini Kit (Qiagen Korea, Seoul, Korea). The dsRNA was confirmed by 1% agarose gel electrophoresis. For bioassays, IPTG-induced recombinant bacterial culture was centrifuged at 7,000 ×g for 10 min. The resulting bacterial cell pellet was resuspended in distilled water. The bacterial suspension was then treated at 95°C for 10 min. Subsequently, the bacterial suspension was subjected to ultrasonication at 100% intensity for 3 cycles (5 second per cycle) with an ultrasonicator (Bandelin Sonoplus, Germany). Quantification of dsRNA amounts produced by recombinant bacteria followed the method described by Kim et al. [30 (link)].
Sonoplus
Manufactured by Bandelin
Sourced in Germany
The Sonoplus is a laboratory equipment designed for ultrasonic homogenization and dispersion of samples. It utilizes high-frequency sound waves to disrupt and mix materials, making it a versatile tool for various applications in research and development laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Sonorex, by Bandelin (5 mentions)
Gm 2200, by Bandelin (2 mentions)
Nanosight ns300, by Malvern Panalytical (2 mentions)
Histrap hp 5 ml column, by GE Healthcare (2 mentions)
Powergen 125, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Nano acquity beh130c18 column, by Waters Corporation (1 mentions)
μc18 ziptip, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
53 protocols using sonoplus
1
Production and Characterization of dsRNA
2
Synthesis of Silicon-Oil-Stabilized Polymer Miniemulsion
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EXAMPLE 15
88.25 g of silicon oil 4 was dissolved in 86.5 g of MMA and 1.77 g of methacrylic acid MAA. The oil phase was added dropwise to a stirred solution of 22.8 g of a solution of surfactant 1 in 130 g of deionised water. . The mixture was stirred for 30 min treated with ultrasound using a Bandelin Sonoplus, Generator GM 2200, 200 W, 20 kHz, at 60% power for 20 minutes per 500 g of emulsion. A kinetically stable miniemulsion was obtained, wherein the average droplet size was 150 nm.
4.4 g of a 10 wt % aqueous solution of t-butyl hydroperoxide was added to the miniemulsion. The miniemulsion was heated to 40° C. To the reactor 18.4 g of a 4.8 wt % aqueous solution of Rongalit C in water was added at 40° C. within 1 hour. The reaction mixture was continuously stirred by a mechanical stirrer and was maintained at 60° C. for 1 hour, then cooled to 22° C. Then the pH of the obtained polymer dispersion was adjusted to 9.1 by addition of 0.48 g of AMP and the dispersion was filtered via a 200 μm filter. The resulting dispersoin had a particle size DINT of 112 nm with a polydispersity of 36%. The final active content (calculated as silicon oil 4) was 25 wt %, the solid content was 52.1 wt %. The residual monomer content (MMA) was 50 ppm. The aspect was a white, slightly viscous (<500 mPas) dispersion.
3
TiO2 Suspension Preparation and Cell Exposure
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The TiO 2 samples were suspended in the culture media described below at a concentration of 0,5 mg/ml in a eppendorf tube. The suspensions were sonicated for 1 min twice with a probe sonicator (Sonoplus, Bandelin, Berlin, Germany) with the following experimental setting:100 W, 20 kHz,attenuation 40%, probe in titanium, diameter of the probe 13mm). The suspension was administered to the cell culture in reduced illumination conditions, i.e. by obscuring the hood to reduce illumination by indoor light thus avoiding photo-activation of the powders.
4
Preparation of GPL Micelle Formulations
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Nutritional Therapeutics, Inc. (Hauppuage, NY, USA) specifically prepared the formulation of NTFactor® Lipids for use in this study, which omitted the fructooligosaccharides from the formulation that are contained within the traditional dietary supplement to protect the GPL against degradation within the digestive system. GPL micelle stock solutions were prepared by adding 3% (w/v) modified NTFactor® Lipids (Nutritional Therapeutics, Inc. of Hauppuage, NY, USA) to BWW. For micelle formation; ethanol (0.1% final volume) was added to the mixture, which was then vortexed until visibly dissolved. The GPL mixture was then placed in an ice bath and ultrasonicated at 20 kHz intermittently for 20 min (5 s on, 5 s off) using a probe sonicator (Bandelin Sonoplus, Germany). The resulting product was filtered through a 0.2 µm sterile syringe filter to isolate sub-micrometre-sized micelles as previously described (Ferreira et al. 2018 (link)).
5
Synthesis of Silicone Oil-based Miniemulsion
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EXAMPLE 12
160 g of silicon oil 4 was dissolved in 12.8 g of MMA, 23.2 g of EHA, 3.2 g of SMA, 0.8 g of MAA and 0.12 g of BDDA. The oil phase was added dropwise to a stirred solution of 25.8 g of surfactant 1 in 163.4 g of deionised water. The mixture was stirred for 30 min treated with ultrasound using a Bandelin Sonoplus, Generator GM 2200, 200 W, 20 kHz, at 60% power for 20 minutes per 500 g of emulsion. A kinetically stable miniemulsion was obtained, wherein the average droplet size was 274 nm.
2.0 g of a 10 wt % aqueous solution of t-butyl hydroperoxide was added to the miniemulsion. The miniemulsion was heated to 40° C. To the reactor 8.3 g of a 4.8 wt % aqueous solution of Rongalit C was added at 40° C. within 1 hour. The reaction mixture was continuously stirred by a mechanical stirrer and was maintained at 60° C. for 1 hour, then cooled to 22° C. Then the pH of the obtained polymer dispersion was adjusted to 9.2 by addition of 1.2 g of AMP and the dispersion was filtered via a 200 μm filter. The resulting dispersoin had a particle size DINT of 199 nm with a polydispersity of 45%. The final active content (calculated as silicon oil 4) was 40 wt %, the solid content was 53 wt %. The residual monomer content (MMA) was <10 ppm. The aspect was a white, low viscous (<100 mPas) dispersion. The glass transition temperature of the polymer matrix was 0° C.
6
Extraction of Nuclear Proteins from OG2 ES Cells
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OG2 ES cells were cultured under feeder-free conditions and washed in phosphate-buffered saline (PBS) before trypsin digestion. Cells were centrifuged at 2500 g for 6 min, and the pellet was washed twice with PBS before it was resuspended in 5 packed cell volumes of prechilled Buffer A (10 mM KCl, 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 2 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 0.2% IGEPAL CA 630). Cells were incubated on ice for 30 min before being centrifuged at 2000 g for 8 min at 4°C. All further work was performed at 4°C unless stated otherwise. The pellet was resuspended in 2 packed cell volumes of Buffer A plus 0.2% IGEPAL, and cell lysis was monitored. After lysis, the sample was centrifuged at 1000 g for 10 min and the supernatant was carefully removed. Nuclei were centrifuged at 4000 g for 10 min, the supernatant was removed, and the cell pellet was resuspended in 1 packed cell volume of RIPA buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 1% IGEPAL CA 630, 0.5% sodium deoxycholate, 1 mM EDTA, 1× protease inhibitors [Roche, Mannheim, Germany], and 1× phosphatase inhibitors [Sigma, St. Louis, MO]). Nuclei were sonicated three times (10 sec, 1-msec pulses, 30% power) using a Bandelin Sonoplus ultrasonic homogenizer. The nuclear extract was centrifuged at 16,000 g for 20 min and the sample was frozen in liquid nitrogen.
7
Purification of GST-Fusion Proteins
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Recombinant glutathione S-transferase (GST) fusion proteins were purified by GST Bind resin (Novagen) following the manufacturer’s instructions. Cells were disrupted by sonication (3min, 5s intervals, 50% intensity; Bandelin Sonoplus, Berlin, Germany) after re-dissolving them in 10ml of binding buffer containing 10 µM of the proteinase inhibitor phenylmethylsulfonyl fluoride. The crude protein extract was incubated for 2h with the resin in order to bind the GST fusion proteins. The recombinant proteins were eluted with GST elution buffer containing 100mM reduced glutathione and quantified (Bradford, 1976 (link)). The identity of the heterologously expressed proteins was verified by SDS–PAGE and western blot (anti-GST antibody).
8
Isolation of Zinc-Solubilizing Bacteria from Soil
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For isolation of Zn-solubilizing bacteria, soil samples were taken from the experimental site. The total five soil samples were taken from 0–20 cm depth and they were made composite sample. One gram of soil was added to 10 ml of 50 mM phosphate buffer (pH 7.0) and 50% of the soil mixture was treated by sonication with an electronic homogenizer (Bandelin Sonoplus, Berlin, Germany) at 260 W/cm2 for 15 secs to isolate bacteria. Serial dilutions were performed after mixing both sonicated and non-sonicated portions. The diluted aliquots were spread on modified half-strength R2A agar plates [24 ]. A 100 μl aliquot was applied to half-strength R2A agar medium in large polystyrene Petri dishes (15 cm diameter) for 10−3 and10-5dilutions, and the plates were incubated at 28°C for 72 hours. The isolation medium was supplemented with 40% (v/v) soil extract and 50 μg/ml amphotericin B to inhibit fungal growth. The colonies were selected based on morphology, and the isolates were sub-cultured on modified half-strength R2A agar plates.
Out of 26 strains, only 4 (PMEL-1, PMEL-48, PMEL-57, and PMEL-71) were selected on the basis of their Zn solubilizing ability and success for 16S rRNA sequencing.
Out of 26 strains, only 4 (PMEL-1, PMEL-48, PMEL-57, and PMEL-71) were selected on the basis of their Zn solubilizing ability and success for 16S rRNA sequencing.
9
Cell Culture Protocols for Diverse Cell Lines
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The human embryonic kidney (HEK) cell lines 293 and 293T, the human cervix carcinoma cell line HeLa, the human melanoma cell line WM35, the human ductal carcinoma cell line MCF-7, the human osteosarcoma cell line U2OS and the colon carcinoma cell line HRT-18 (also termed HCT-8) were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, United States) supplemented with 10% FBS (PAA) plus 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, United States) according to ATCC guidelines. CCRF-CEM-C7H2 (CEM) is human acute leukemic cell line glucocorticoid-sensitive subclone of CCRF-CEM (Strasser-Wozak et al., 1995 (link)) and HEL is a human erythroleukemia cell line (Martin and Papayannopoulou, 1982 (link); Jäkel et al., 2011 (link)). These cell lines were cultured in RPMI-1640 (Sigma-Aldrich). The mouse cardiac muscle cell line HL-1 was cultured in Claycomb media.
A total of 293 and 293T cells were transfected by calcium-phosphate precipitation (Graham and van der Eb, 1973 (link)), HRT-18 cell by Polyfect or Lipofectamine 2000 (Thermo Fisher Scientific). Cells were lysed in Laemmli buffer (Laemmli, 1970 (link)) or IP-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40 and protease inhibitor cocktail (Sigma Aldrich, St Louis, MO, United States) using an ultrasonic homogenizer (Sonoplus, Bandelin, Berlin, Germany).
A total of 293 and 293T cells were transfected by calcium-phosphate precipitation (Graham and van der Eb, 1973 (link)), HRT-18 cell by Polyfect or Lipofectamine 2000 (Thermo Fisher Scientific). Cells were lysed in Laemmli buffer (Laemmli, 1970 (link)) or IP-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40 and protease inhibitor cocktail (Sigma Aldrich, St Louis, MO, United States) using an ultrasonic homogenizer (Sonoplus, Bandelin, Berlin, Germany).
10
Necrotic Cell Lysate Preparation
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Cells suspended at 4 × 106 cells/mL DMEM were subjected to (i) sonication three times at 15 s intervals (Sonoplus; Bandelin electronic GmbH & Co. KG; Berlin, Germany) or (ii) three times freeze–thawing for 8 min at −80 °C and room temperature. Cell lysates underwent centrifugation at 2600 RCF for 5 min (Eppendorf SE, Hamburg, Germany). Necrotic cell lysates were freshly prepared for each independent test. In brief, gingival fibroblasts were seeded at 30.000 cells/cm2 and on the following day exposed to the undiluted cell lysates for another 24 h before analysis.
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