T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs. DNA Polymerase Mastermix was from Denville Scientific. Ni-NTA Superflow, QIAprep Miniprep kits, and QIAquick gel extraction kits were purchased from Qiagen. All reagents and cofactors were from Sigma-Aldrich or Thermo Fisher Scientific. Oligonucleotide primers were synthesized by Valuegene and IDT. Gene sequencing and gene synthesis were performed by Genewiz and Twist Bioscience. Assembly master mix (AMM) used for cloning was prepared as outlined in19 (link). All DNA and protein concentrations were measured with a Thermo Fisher Scientific Nanodrop 1000 Spectrophotometer. Vials for small scale reactions, for gas chromatography and 96-well plates were purchased from VWR. Viton tubing was purchased from Cole-Parmer Instrument Company. Colorimetric enzyme-coupled assays were performed in 96-well plates and measured with Molecular Devices SpectraMax M5 microplate reader. When performing large numbers of assays simultaneously, Integra Viafill and Integra Viaflo instruments were used for reagent additions. Colonies for screening of mutant variants were selected automatically using a Molecular Devices Qpix 420 colony picker.
Viaflo
Manufactured by Integra LifeSciences
Sourced in United States
The Viaflo is a compact, handheld electronic pipette designed for liquid handling tasks in the laboratory. It offers adjustable volume ranges and supports a variety of pipette tips for accurate and precise volume delivery.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qiaprep miniprep kit, by Qiagen (1 mentions)
Dna polymerase mastermix, by Thomas Scientific (1 mentions)
Viafill, by Integra LifeSciences (1 mentions)
T4 dna ligase and restriction endonucleases, by New England Biolabs (1 mentions)
Ni nta superflow, by Qiagen (1 mentions)
Spectramax m5 microplate reader, by Molecular Devices (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Viton tubing, by Cole-Parmer (1 mentions)
Anti dykddddk tag monoclonal antibody, by Fujifilm (1 mentions)
Nanohead, by PerkinElmer (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Express five sfm medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Falcon 96 well tissue culture plates, by Corning (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Tissue culture flask, by Corning (1 mentions)
Half area black plate, by Corning (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
6 protocols using viaflo
1
High-Throughput Molecular Biology Workflow
2
High-Throughput Protein Interaction Screening
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All AlphaScreen reactions were conducted in AlphaPlate 384 titer plates (PerkinElmer). 20 ml of solution containing 0.5 μl of a biotin-tagged bait protein and 19.5 μl of the AlphaScreen buffer was dispensed into reaction plate by using automated multichannel pipet Viaflo and Viaflo Assist system (Integra). Next, 1 μl of each FLAG-GST tagged protein array protein was transferred from 384-well stock plate to the reaction plate using Janus automated dispensing workstation (PerkinElmer) and Nanohead, a 384-well micro syringe head (PerkinElmer). Then 9 μl of detection mixture containing 0.02 μl of anti-DYKDDDDK tag monoclonal antibody (Wako), 0.06 μl of streptavidin-conjugated AlphaScreen donor beads and 0.06 μl of protein A–conjugated AlphaScreen acceptor beads (PerkinElmer) in the AlphaScreen buffer were added to the reaction plates using FlexDrop dropper. After incubation at 25°C for 1 h in a dark incubator, AlphaScreen signal was detected by Envision multilabel reader (PerkinElmer).
3
Drosophila Melanogaster S2 Cell Cultivation
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Drosophila melanogaster S2 cells were firstly thawed at passage 12 with Schneider's Drosophila Medium (Bio&Sell, supplemented with 10% FBS (Fetal Bovine Serum, Biochrom)) and later cultivated in Express Five SFM medium (protein‐free and serum‐free, Invitrogen). One bottle of the Express Five medium (1 l) was supplemented with 90 ml of L‐Glutamine (200 mM, Invitrogen). During cultivation, cells were grown at 25°C without CO2 in tissue culture flasks (75 cm2, Corning) and were split into fresh flasks when 90% confluent. The cells in passage 18 were seeded into 96‐well plates (Falcon 96 Well Tissue Culture Plates, Corning) with 40,000 cells per well in 100 µl using an electronic multichannel pipette VIAFLO (1,250 µl, INTEGRA) on a pipetting robot ASSIST (INTEGRA). The cells 24 h growth rate and viability were monitored in the culture dishes (in duplicate; 100 mm, Corning) with 12 × 106 cells in 14 ml medium. Cell counting and assessment of cell viability were performed using the Cell Counter and Analyzer System (CASY, Roche).
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Drosophila melanogaster S2 cells were firstly thawed at passage 12 with Schneider's Drosophila Medium (Bio&Sell, supplemented with 10% FBS (Fetal Bovine Serum, Biochrom)) and later cultivated in Express Five SFM medium (protein‐free and serum‐free, Invitrogen). One bottle of the Express Five medium (1 l) was supplemented with 90 ml of L‐Glutamine (200 mM, Invitrogen). During cultivation, cells were grown at 25°C without CO2 in tissue culture flasks (75 cm2, Corning) and were split into fresh flasks when 90% confluent. The cells in passage 18 were seeded into 96‐well plates (Falcon 96 Well Tissue Culture Plates, Corning) with 40,000 cells per well in 100 µl using an electronic multichannel pipette VIAFLO (1,250 µl, INTEGRA) on a pipetting robot ASSIST (INTEGRA). The cells 24 h growth rate and viability were monitored in the culture dishes (in duplicate; 100 mm, Corning) with 12 × 106 cells in 14 ml medium. Cell counting and assessment of cell viability were performed using the Cell Counter and Analyzer System (CASY, Roche).
4
Disrupting Biofilms with NEBB
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The treatment of established biofilm with NEBB and the resulting disruption of biofilm included release of planktonic forms into the culture supernatant and detachment of clumps of bacteria living in biofilm. In order to provide conclusive data on the effects on biofilms, following published methodology [62 (link)], planktonic forms had to be removed from the cultures before staining for biofilm mass and metabolic activity, The removal of planktonic forms and the addition of washing buffer was done using a very low speed to avoid mechanical removal of biofilm material. The removal of planktonic forms was performed using electronic 12-channel pipettes (Viaflo, Integra, USA), where the speed was set to “1” (the maximum speed is “10”). Phosphate-buffered saline was added, also using speed “1”, where the liquid was dispensed onto the sidewalls of each well to avoid disruption of biofilm by direct pipetting actions onto biofilm. For the cultures of Pseudomonas aeruginosa, this pipetting allowed scoring of slime formation, where “0” indicated no change in viscosity of the culture medium, and a score of “3” (300%) indicated that the entire culture medium had turned into a mucus plug.
5
Pipette-based Biofilm Disruption and Quantification
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The treatment of established biofilm with NEBB and the resulting disruption of biofilm included the release of planktonic forms into the culture supernatant and the detachment of clumps of bacteria living in biofilm. In order to provide conclusive data on the effects on biofilms, following published methodology [37 (link)], planktonic forms had to be removed from the cultures before staining for biofilm mass and metabolic activity. The removal of planktonic forms and the addition of washing buffer were performed using a very low speed to avoid mechanical removal of biofilm material. The removal of planktonic forms was performed using electronic 12-channel pipettes (Viaflo, Integra, Hudson, NH, USA), where the speed was set to “1” (the maximum speed is “10”). Phosphate-buffered saline was added, also using speed “1”, where the liquid was dispensed onto the sidewalls of each well to avoid disruption of biofilm by direct pipetting actions onto biofilm. For the cultures of Pseudomonas aeruginosa, this pipetting allowed the scoring of slime formation, where “0” indicated no change in viscosity of the culture medium, and a score of “3” (300%) indicated that the entire culture medium had turned into a mucus plug.
6
Fluorescence Polarization Assay for MSUT2
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