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.
Viafill
Manufactured by Integra LifeSciences
The Viafill is a versatile automated liquid handling system designed for a wide range of laboratory applications. It features precise pipetting, plate handling, and liquid dispensing capabilities, enabling efficient and accurate sample preparation and processing.
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)
T4 dna ligase and restriction endonucleases, by New England Biolabs (1 mentions)
Viaflo, by Integra LifeSciences (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)
Echo 550, by Beckman Coulter (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
3 protocols using viafill
1
High-Throughput Molecular Biology Workflow
2
High-Throughput Screening of Small Molecule Library for PLpro Inhibitors
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A compound library consisting of 35,360 structurally diverse small molecules was purchased from Chemdiv and maintained as 5 mg/ml stock solutions dissolved in DMSO and stored at -20°C. The automated liquid dispensing of primary screen was performed on Echo 550 (Labcyte) and Viafill (Integra). Fluorescence values were measured on Enspire Multimode Plate Reader (PerkinElmer). All assays were performed in duplicate at room temperature, using black flat-bottom 384-well plates (Greiner Bio-One, #784076) containing a final reaction volume of 20 μL. The assays were assembled as follows: 20 nL compounds (final concentration 5 μg/ml) was dispensed into wells and then incubated with 10 μL of 2 μM PLpro (final concentration 1 μM) in Buffer A (20 mM Tris-HCl, 100 mM NaCl, 5 mM 2-hydroxy-1-ethanethiol, pH 7.8) for 30 min. Reactions were initiated with 10 μL of 5 μM probe 5 (final concentration 2.5 μM) in Buffer A, shaken vigorously for 10 sec, and then incubated for 60 min, before measuring fluorescence emission intensity (λex = 405 nm; λem = 450 nm). Each 384-well plate contained 11 positive control wells (20 nL of DMSO replacing 20 nL of inhibitor in DMSO), 12 negative control wells (assay components lacking PLpro) and 1 positive inhibitor control well (20 nL of 10 mM GRL0617). Compounds that showed Z-scores of less than -8 were cherry picked for further analysis.
3
Nematode Growth Medium Preparation
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Low peptone (0.013%) nematode growth medium (a version of the protocol is maintained at protocols.io https://dx.doi.org/10.17504/protocols.io.2rcgd2w ) was prepared as follows: 20 g agar (Difco), 0.13 g bactopeptone, and 3 g NaCl were dissolved in 975 ml of Milli‐Q water. After autoclaving, 1 ml of 10 mg/ml cholesterol was added along with 1 ml of 1 M CaCl2, 1 ml 1 M MgSO4, and 25 ml of 1 M KPO4 buffer (pH 6.0). Molten agar was cooled to 50–60°C, and 200 μl was dispensed into each well of 96‐square well plates (WHAT‐7701651) using an Integra VIAFILL (a version of the protocol is maintained at protocols.io https://dx.doi.org/10.17504/protocols.io.bmxbk7in ). Poured plates were stored agar side up at 4°C until required.
Prior to applying compounds, plates were placed without lids in a drying cabinet to lose 3–5% weight by volume and then stored with lids (WHAT‐77041001) at room temperature until required.
Prior to applying compounds, plates were placed without lids in a drying cabinet to lose 3–5% weight by volume and then stored with lids (WHAT‐77041001) at room temperature until required.
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