EVs were isolated from K pneumoniae as previously described.[12 (link)] Bacteria were incubated in Luria–Bertani broth at 37°C with shaking at 200 rpm. A bottle top vacuum filter with a pore size of 0.45 μm (Corning, Inc., Corning, NY) was used to obtain the supernatant, which was then filtered using the QuixStand Benchtop System (Amersham Biosciences, Little Chalfont, UK). A bottle top vacuum filter with a pore size of 0.22 μm (Corning, Inc.) was used to remove any residual bacteria from the supernatant. EVs from K pneumoniae were harvested and confirmed by transmission electron microscopy (TEM). Images of the EVs were taken via a Hitachi (H7650, 80 kV) TEM (Fig. 1 ).
Bottle top vacuum filter
Manufactured by Corning
Sourced in United States
The Bottle Top Vacuum Filter is a laboratory equipment designed for the filtration of liquids. It is used to separate particulate matter from a liquid solution through the application of vacuum pressure. The device consists of a filter unit that is attached to the top of a collection bottle, allowing the filtered liquid to be collected for further use or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quixstand benchtop system, by Cytiva (1 mentions)
H 7650, by Hitachi (1 mentions)
Ribopure yeast, by Thermo Fisher Scientific (1 mentions)
Pes membrane, by Corning (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Qiagen (1 mentions)
Follicle growth medium kit, by PromoCell (1 mentions)
Hdpcs, by PromoCell (1 mentions)
Alliin, by MedChemExpress (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
Quixstand benchtop system, by GE Healthcare (1 mentions)
10 protocols using bottle top vacuum filter
1
Extracellular Vesicle Isolation from Klebsiella pneumoniae
2
Recombinant Xylanase Production in Aspergillus oryzae
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Example 28
The xylanase with nucleotide sequence SEQ ID NO: 97 was PCR amplified from genomic DNA isolated from Lasiodiplodia theobromae and cloned into the expression vector pSUN515, which is a derivative of pCaHj505 (WO 2013/029496).
The final expression plasmid was transformed into the Aspergillus oryzae MT3568 expression host. A. oryzae MT3568 is a derivative of A. oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene. The xylanase gene was integrated by homologous recombination into the A. oryzae MT3568 host cell genome upon transformation.
The gene coding for amdS was used as marker. Transformants were selected on pyrG media agar supplemented with 10 mM acetamide. One recombinant Aspergillus oryzae clone containing the xylanase expression construct was selected and was cultivated on a rotary shaking table in 4 2-liter baffled Erlenmeyer flasks each containing 400 ml YPM (1% Yeast extract, 2% Peptone and 2% Maltose). After 3 days cultivation time at 30° C., enzyme containing supernatants were harvested by filtration using a 0.22 μm 1-liter bottle top vacuum filter (Corning Inc., Corning, N.Y., USA).
3
Cloning and Expression of Xylanase from Lasiodiplodia theobromae
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Example 28
The xylanase with nucleotide sequence SEQ ID NO: 97 was PCR amplified from genomic DNA isolated from Lasiodiplodia theobromae and cloned into the expression vector pSUN515, which is a derivative of pCaHj505 (WO 2013/029496).
The final expression plasmid was transformed into the Aspergillus oryzae MT3568 expression host. A. oryzae MT3568 is a derivative of A. oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene. The xylanase gene was integrated by homologous recombination into the A. oryzae MT3568 host cell genome upon transformation.
The gene coding for amdS was used as marker. Transformants were selected on pyrG media agar supplemented with 10 mM acetamide. One recombinant Aspergillus oryzae clone containing the xylanase expression construct was selected and was cultivated on a rotary shaking table in 4 2-liter baffled Erlenmeyer flasks each containing 400 ml YPM (1% Yeast extract, 2% Peptone and 2% Maltose). After 3 days cultivation time at 30° C., enzyme containing supernatants were harvested by filtration using a 0.22 μm 1-liter bottle top vacuum filter (Corning Inc., Corning, N.Y., USA).
4
Isolation and Purification of dsRNA from T. marneffei
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To isolate dsRNA, 10 ml cultures of the 55 T. marneffei isolates in the mycelial phase were filtered using a Corning bottle top vacuum filter (150 ml) with a pore size of 0.22 µm and a polyethersulfone (PES) membrane (Corning, Corning, NY), and the mycelial mass was collected and subjected to total RNA extraction using RiboPure yeast (Ambion, USA) as described previously (31 (link), 33 (link)) followed by treatment with 8 units of DNase I (Ambion)–5 µg/µl RNase A (Qiagen, Hilden, Germany)–2× SSC buffer (Ambion; 1× SSC buffer is 0.3 M NaCl, 30 mM sodium citrate) for 1 h at 37°C, separated by electrophoresis through 1% agarose gels, and visualized under UV light after staining with ethidium bromide, as described previously (79 (link), 80 (link)). The isolated dsRNA was purified using a PureLink RNA minikit (Ambion) and stored at −80°C before use.
5
Extraction and Characterization of Allium hookeri
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HDPCs provided by PromoCell (Heidelberg, Germany) were purchased and maintained in a follicle growth medium kit (Promocell, Heidelberg, Germany) with 5% CO2 and at 37 °C. Passage 4 to 6 HDPCs were used in in vitro cultivation and all the experiments in this study. Allium hookeri used in this study were collected in March 2022 from a cultivation area located in the Pyeongchang region (Gangwon-do, Republic of Korea). The plants were washed with distilled water three times and air-dried at room temperature (in the shade). Fifty grams of dried A. hookeri was chopped and ground into a powder using a grinder (SMX-3500GN, Shinil Industrial Co., Ltd., Seoul, Republic of Korea). Then, 50 g of the ground A. hookeri powder was mixed with 900 mL of distilled hot water (80 °C) for 4 h. The obtained extracts were filtered through Whatman filter paper No. 1 (Whatman, Maidstone, UK) followed by ultrafiltration through a sterile 0.2 µm bottle-top vacuum filter (Corning, Corning, NY, USA). Using HPLC-HRMS and DPPH analyses, the extract was determined to have the main component, alliin (MedChemExpress, Monmouth Junction, NJ, USA), and a radical scavenging effect; therefore, these two methods were used to monitor the quality of the extracts.
6
Cloning and Expression of Xylanase from Lasiodiplodia theobromae
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Example 28
The xylanase with nucleotide sequence SEQ ID NO: 97 was PCR amplified from genomic DNA isolated from Lasiodiplodia theobromae and cloned into the expression vector pSUN515, which is a derivative of pCaHj505 (WO2013029496).
The final expression plasmid was transformed into the Aspergillus oryzae MT3568 expression host. A. oryzae MT3568 is a derivative of A. oryzae JaL355 (WO02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene. The xylanase gene was integrated by homologous recombination into the A. oryzae MT3568 host cell genome upon transformation.
The gene coding for amdS was used as marker. Transformants were selected on pyrG media agar supplemented with 10 mM acetamide. One recombinant Aspergillus oryzae clone containing the xylanase expression construct was selected and was cultivated on a rotary shaking table in 4 2-liter baffled Erlenmeyer flasks each containing 400 ml YPM (1% Yeast extract, 2% Peptone and 2% Maltose). After 3 days cultivation time at 30° C., enzyme containing supernatants were harvested by filtration using a 0.22 μm 1-liter bottle top vacuum filter (Corning Inc., Corning, N.Y., USA).
7
Isolation of Staphylococcus aureus Extracellular Vesicles
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Staphylococcus aureus, one of the bacteria listed in Table 1 , was selected for the experiments. A strain of S. aureus, purchased from the American Type Culture Collection (ATCC 14458), and grown in Luria-Bertani broth at 37 °C and 200 rpm. A top-bottom vacuum filter (Corning, NY, USA) with a pore size of 0.45 μm and a QuixStand Benchtop System (GE Healthcare, Chicago, IL, USA) was used for filtration. Residual bacteria were removed from the supernatant using a bottle top vacuum filter with a pore size of 0.22 µm (Corning, NY, USA). The filtered extraction was ultracentrifuged at 150,000× g for 3 h at 4 °C on a type 45 Ti rotor (Beckman Instruments, Brea, CA, USA). After this step, the EV pellets were obtained and resuspended in PBS. The extracted EVs were stored at −80 °C [29 (link)].
8
Quantitative Serum MMA Assay Protocol
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Pooled serum was filtered using a disposable Corning bottle-top vacuum filter with a 0.22 μm membrane and aliquoted. Four pooled samples with different concentrations of MMA were prepared for the precision and recovery evaluation, that is, sample pools (Level 1), low- (Level 2), medium- (Level 3), and high-levels (Level 4) of QC materials described before. All samples were aliquoted into 2.0 mL Corning vials (1 mL/vial) and stored at −40 °C before analysis. Each sample was measured five times per day for five days.
9
Wastewater Nutrient Removal Capacity Analysis
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The nutrient removal capacity were analysed on beginning (0 day) and end (10 day) of the experiment by measuring physico-chemical parameters such as total nitrogen, total phosphate, COD, BOD, TOC, TIC, iron magnesium, alkalinity and hardness. These parameters were studied following standard methods reported by the American Public Health Association (APHA). 26 On beginning of the experiment, wastewater was filtered using 0.2µm filters (Corning® bottle-top vacuum filters) and filtrates were analysed for physic-chemical parameter. On end of the experiment (10 th ), algal biomass was harvested by centrifugation at 3000 rpm for 15 min at 4 o C as reported, 27 and remaining wastewater (supernatant) was filtered using 0.2µm filters. Then, filtrates were subjected to physico-chemical analysis.
The percentage removal of nutrients was calculated using the following equation:
where, C o and C F stand for initial concentration on beginning of the experiment (0 day) and final concentration on end of experiment (10 day), respectively.
The percentage removal of nutrients was calculated using the following equation:
where, C o and C F stand for initial concentration on beginning of the experiment (0 day) and final concentration on end of experiment (10 day), respectively.
10
Neela Hauze Lake Wastewater Filtration
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Wastewater was collected in bulk from the Neela Hauze Lake situated between 28.528950° N latitude and 77.170910° E longitude, New Delhi. Sedimentation and filtration through 0.2µm filters (Corning® bottle-top vacuum filters) removed solid particles. After filtration, wastewater was stored at 4°C in the dark until needed for the experiments.
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