Tissue samples from the caudal part of the caecum were cut into 20 mg pieces, placed immediately into RNA Later solution (Qiagen, UK), and stored at −70°C prior to RNA purification. Single tissue fragments were transferred into 1 ml of TRIzol Reagent (Molecular Research Center, USA), and homogenized with zirconium silica beads (BioSpec Products, USA) in a vortex mixer (Labnet, USA). To separate the phases, 50 μl of 4-bromanisole (Molecular Research Center, USA) was added. The whole content of the tube was centrifuged and the upper aqueous phase was collected for total RNA purification with the RNAeasy mini kit (Qiagen, UK), according to the manufacturer's instructions. Turbo DNA-free kit (Ambion, USA) was used for the treatment of RNA samples to remove genomic DNA. Both the purity and concentration of RNA were determined spectrophotometrically on NanoDrop 200c (Thermo Scientific, USA) and 1 μg of the total RNA immediately underwent reverse transcription with iScript cDNA Synthesis Kit (Bio-Rad, USA). The resulting cDNA was diluted 10-fold in UltraPure DNase/RNase-Free distilled water (Invitrogen, USA) and used as a template for real-time PCR, or stored at −20°C until used.
Vortex mixer
Manufactured by Labnet
Sourced in United States
The Vortex mixer is a laboratory equipment used for mixing and agitating various types of samples. It creates a vortex motion to thoroughly mix the contents of a tube or container.
Automatically generated - may contain errors
Lab products found in correlation
Zirconium silica beads, by Biospec (1 mentions)
Nanodrop 200c, by Thermo Fisher Scientific (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
4 bromanisole, by Molecular Research Center (1 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Trizol reagent, by Molecular Research Center (1 mentions)
Rnalater solution, by Qiagen (1 mentions)
H 7650, by Hitachi (1 mentions)
Cm0007, by Thermo Fisher Scientific (1 mentions)
4 protocols using vortex mixer
1
Caecum Tissue RNA Extraction Protocol
2
Atmospheric Cold Plasma Treatment of Wheat Flour
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The ACP equipment used in this study is shown in Fig. 1 . A certain amount (0.5 to 1.5 g) of wheat flour was transferred into a 9.5-ml customized polypropylene container. The distance between the top and bottom of the customized container was 30 mm. Also, the distance from the flour surface to the tip of the plasma wand was 10-20 mm. The container was placed on a vortex mixer (Labnet Inc., USA) and the flour particles were continuously vibrated at 3,400 rpm for efficient contact between the flour particles and the plasma. The gas nozzle connected to the gas cylinder through the gas flow meter was connected to the 30 W plasma generator (Plasma Etch Inc., USA) that was fixed to the retort stand. The temperature of the ACP was under 50°C, and the atmospheric conditions were 20 ± 2°C and 22 ± 2% relative humidity, as measured by a hygrometer.
3
Yeast Cell Lysate Preparation and Protein Purification
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Cell lysates were prepared as described previously [11] , with slight modifications. Yeast cultures (30 ml) were harvested by centrifugation at 1,500 ×g for 5 min, resuspended in 15 ml of disruption buffer (10 mM NaH 2 PO4, 150 mM NaCl, 1.7 mM ethylenediaminetetraacetic acid, and 0.01% Tween 80, pH 7.2), mixed with 0.5 mm diameter glass beads (15 g), and disrupted using a vortex mixer (Labnet International, Inc., USA) for 30 min. Cell debris were removed by centrifugation at 1,500 ×g for 5 min. The supernatants were re-centrifuged at 12,000 ×g for 10 min, collected, and precipitated using 40% saturated (NH 4 ) 2 SO 4 for 30 min at room temperature. The precipitated samples were collected by centrifugation at 12,000 ×g for 10 min, washed twice in 1 ml of 20% saturated (NH 4 ) 2 SO 4 using a pipette, collected by centrifugation at 12,000 ×g for 10 min, and dissolved in 100 µl of disruption buffer.
The samples were absorbed on carbon-coated copper grids and negatively stained with 2% phosphotungstic acid. The grids were air dried prior to examination under a transmission electron microscope (Hitachi, H-7650) with 80 kV acceleration voltage [9] .
The GenBank accession number of the sequence that is translated to HPV type 52 late protein L1 reported in this paper is KJ778071.1.
The samples were absorbed on carbon-coated copper grids and negatively stained with 2% phosphotungstic acid. The grids were air dried prior to examination under a transmission electron microscope (Hitachi, H-7650) with 80 kV acceleration voltage [9] .
The GenBank accession number of the sequence that is translated to HPV type 52 late protein L1 reported in this paper is KJ778071.1.
4
Biosensor Calibration Curve Experiments
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Raw milk used for the definition of calibration curves experiments was collected aseptically from healthy cows. Conventional microbiological tests were performed according to NMC protocols [16 ], to confirm no bacterial growth. Briefly, a raw milk sample (10 μL) was plated on a Columbia agar plate and on MacConkey agar plate (CM0007, Oxoid, Hampshire, UK) and both were incubated at 37 °C for 48 h. The absence of growth on both plates was considered to be equivalent to the presence of no viable bacteria in the milk.
Each sample for biosensor testing had a 500 µL volume consisting of 2 µL of a suspension of functionalized NPs, 98 µL of PBST, and 400 µL of one of seven bacterial suspensions with pre-defined bacterial concentrations in PBS or sterile raw milk. The incubation of these samples was performed at RT for 3 h, under agitation.
All raw milk samples were submitted to a pre-treatment of 15 min at 60 °C in a dry bath incubator (Grant, model QBD2, Leicestershire, UK) and 15 min of continuous centrifugation in a vortex mixer (Labnet International Inc., Edison, NJ, USA) after adding bacteria and PBST. Only then, 2 µL of functionalized NPs suspension were added for final incubation step.
Each sample for biosensor testing had a 500 µL volume consisting of 2 µL of a suspension of functionalized NPs, 98 µL of PBST, and 400 µL of one of seven bacterial suspensions with pre-defined bacterial concentrations in PBS or sterile raw milk. The incubation of these samples was performed at RT for 3 h, under agitation.
All raw milk samples were submitted to a pre-treatment of 15 min at 60 °C in a dry bath incubator (Grant, model QBD2, Leicestershire, UK) and 15 min of continuous centrifugation in a vortex mixer (Labnet International Inc., Edison, NJ, USA) after adding bacteria and PBST. Only then, 2 µL of functionalized NPs suspension were added for final incubation step.
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