Phage lysates were purified on a cesium chloride gradient by ultracentrifugation [25 (link)], and 100 μl were dialyzed against phage buffer for 20 min on 0.025 μm VSWP membrane filters (Merck Millipore). Negative staining of phages and transmission electron microscopic analysis were performed as previously described [29 (link)].
Vswp membrane filter
Manufactured by Merck Group
Sourced in Germany
The VSWP membrane filters are laboratory filtration devices used for the filtration of aqueous solutions. They are made of mixed cellulose esters and have a pore size of 0.025 μm. These filters are designed to effectively remove particulates, microorganisms, and other contaminants from liquid samples.
Automatically generated - may contain errors
7 protocols using vswp membrane filter
1
Phage Purification and Characterization
2
Phage Purification and Negative Staining
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Phage cultures purified by CsCl gradient centrifugation were dialyzed against phage buffer (20 mM Tris-HCl pH 7.2, 10 mM NaCl, 20 mM MgSO4) by 20 min microdialysis on 0.025 μm VSWP membrane filters (Merck Millipore, Darmstadt, Germany). Ultrathin carbon films (~3 × 3 mm in size) were floated from mica-sheets into a drop of phage culture (100 μL, 2 × 1010 PFU/mL), and after an adsorption time of 20 min, samples were transferred into a drop of 1% (vol/vol) of electron-microscopy-grade glutaraldehyde (20 min) for fixation. Negative staining with 2% uranyl acetate and subsequent transmission electron microscopy (TEM) (Tecnai 10; FEI Thermo Fisher Scientific) was done as described earlier [24 (link)] at an acceleration voltage of 80 kV. Micrographs were captured with a MegaView G2 charge-coupled device camera (Emsis, Muenster, Germany).
3
Phage Purification and Negative Staining
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Phages purified by CsCl gradient centrifugation were dialyzed against phage buffer [20 mM Tris-HCl (pH 7.2), 10 mM NaCl, 20 mM MgSO4] [20-min microdialysis on 0.025 μm VSWP membrane filters (Merck Millipore, Darmstadt, Germany)]. Ultrathin carbon films (~3 × 3 mm in size) were floated from mica-sheets into a drop of phage lysate (100 μL), and after an adsorption time of 20 min, samples were transferred into a drop of 1% (vol/vol) of electron microscopy grade glutaraldehyde (20 min) for fixation. Negative staining with 2% uranyl acetate and subsequent transmission electron microscopy (TEM) (Tecnai 10; FEI Thermo Fisher Scientific, Eindhoven, the Netherlands) was done as described earlier at an acceleration voltage of 80 kV (32 (link)). Micrographs were captured with a MegaView G2 charge-coupled device camera (Emsis, Muenster, Germany).
4
Dialysis Assay for Denatured FilP
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For each dialysis assay, denatured FilP in 6 or 8 M urea was dialyzed against the favored buffer (see below). The dialysis was performed on 0.025 μm VSWP membrane filters (Millipore) at 4°C for 1 h in 20-ml plastic Petri dishes containing dialysis buffer.
5
Plasmid-Mediated Expression of WspR9 in WSs
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Plasmids pVSP61-WspR9 and pVSP61 (control) were used to assess the impact of the expression of WspR9 (Goymer et al. 2006) in representative WSs. Plasmid DNA was isolated using a Gene-Jet Plasmid Miniprep Kit (Thermo Scientific, UK) and dialysed against deionized water for 1 h using 0.025 μm VSWP membrane filters (Millipore, UK). Aliquots of cells (100 μL) were prepared from overnight KB cultures, washed twice and then resuspended in an equal volume of ice-cold 10% (v/v) glycerol, 1 mM HEPES solution. Freshly prepared cells were electroporated with 10 μL DNA in ice-cold 1 mm gap-width cuvettes using an Electroporator 2510 (Eppendorf, UK) set at 200 , 1.75 kV and 25 μF. Cells were outgrown in 1 mL KB for 1 h at 28 • C before aliquots were spread on KB plates containing 50 μg mL -1 kanamycin and incubated for 48 h at 28 • C. Transformant colonies were restreaked onto fresh selective plates and stored at -80 • C as for the WS isolates. Attachment levels were determined as for the wrinkleality assay in static microcosms containing 50 μg mL -1 kanamycin. Repression of attachment was calculated as A 570 pVSP61 strain/A 570 pVSP61-WspR strain.
6
MALDI-TOF Analysis of Cas13a-Processed pre-crRNA
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Processed pre-crRNA samples for MALDI experiments were obtained by incubation of 3′-FAM-labeled pre-crRNA or respective desoxy-modified pre-crRNA (dA-modified/dC-modified) (24 µM; or 15 µM; Ella Biotech; sequences in Supplementary Table S2 ) with RcCas13a (33 µM) in cleavage buffer for 3 h at 37 °C. The Cas13a protein was removed by heat denaturation (95 °C; 10 min) followed by centrifugation (16,000 × g; 10 min; 4 °C. Unprocessed pre-crRNA (100 µM in H2O) served as control. MALDI-TOF mass spectra were recorded on a Bruker Autoflex II. For the measurements, the samples were desalted against ddH2O using a 0.025 μm VSWP membrane filter (Merck Millipore) and then co-crystallized with a matrix solution (0.7 M 3-hydroxypicolinic acid, 0.07 M diammonium citrate in 1:1 H2O/MeCN).
7
MALDI Analysis of Cas13a-Processed pre-crRNA
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Processed pre-crRNA samples for MALDI experiments were obtained by incubation of 3'-FAMlabeled pre-crRNA (24 µM; 72 pmol) with RcCas13a (33 µM) in cleavage buffer for 3 h at 37 °C. The Cas13a protein was removed by heat denaturation (95°C; 10 min) followed by centrifugation (16 000 x g; 10 min; 4°C). Unprocessed pre-crRNA (100 µM in H2O) served as control. MALDI-TOF mass spectra were recorded on a Bruker Autoflex II. For the measurements, the samples were desalted against ddH2O using a 0.025 μm VSWP membrane filter (Merck Millipore) and then co-crystallized with a matrix solution (0.7 M 3-hydroxypicolinic acid, 0.07 M diammonium citrate in 1:1 H2O/MeCN).
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