The flowering buds of each plant were collected and dried at 40 °C for at least 12 h and subsequently ground in an electric mill (IKA® Tube Mill Control, Staufer, Germany). Then, 100 ± 10 mg of dry homogenized herbal Cannabis was automatically extracted using the Tecan Freedom Evo 150 liquid handling platform (Tecan Trading, Männedorf, Switzerland). The extraction procedure was carried out by adding 10.0 mL of 99% denaturated ethanol (VWR BDH Prolabo Chemicals, Leuven, Belgium)—containing 0.01 mg/mL of internal standard, tribenzylamine (Alfa Aesar, Karlsrühe, Germany)—to each sample. Subsequently, the Tecan® instrument sonicated all samples for 15 min, followed by 5 min of horizontal shaking (100 turns). Afterwards, 1.0 mL of the obtained extracts was collected twice in 1 mL vials and subjected to one of the chromatographic methods.
Freedom evo 150
Manufactured by Tecan
Sourced in Switzerland, Austria, United States
The Freedom EVO 150 is a liquid handling workstation designed for laboratory automation. It features a robotic arm and various peripherals to automate liquid transfer and sample processing tasks. The core function of the Freedom EVO 150 is to provide automated liquid handling capabilities to increase efficiency and reproducibility in laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Maxwell simplyrna kit, by Promega (2 mentions)
Paxgene blood rna kit, by Qiagen (2 mentions)
Cfx384 instrument, by Bio-Rad (2 mentions)
Hard shell 384 well microplate, by Bio-Rad (2 mentions)
Microseal b clear, by Bio-Rad (2 mentions)
Yeast trna, by Roche (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
96 well round bottom plate, by Corning (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
El406 plate washer, by Agilent Technologies (2 mentions)
250 nl pin transfer tool, by V&P Scientific (2 mentions)
Flexmap 3d instrument, by DiaSorin (2 mentions)
Nephelostar plus, by BMG Labtech (1 mentions)
Software, by FlowJo (1 mentions)
Evo 150, by Tecan (1 mentions)
Rneasy plus mini extraction kit, by Qiagen (1 mentions)
Taqman primer probe set, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Bioactive compound library, by Topscience (1 mentions)
40 protocols using freedom evo 150
1
Automated Extraction and Analysis of Cannabis
2
Quantification of Bioactive Compounds
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The flowering buds were dried in an oven at 40°C for at least 12 hours. The herbal material was grinded using an IKA ® Tube Mill Control (IKA, Staufer, Germany) and afterwards homogenized and accurately weighed (100 ± 10 mg) into 12-ml glass test tubes. The weighed samples were subjected to the extraction step, applying the Tecan Freedom Evo 150 liquid handling platform (Tecan Trading AG, Männedorf, Switzerland). This instrument adds 10.0 ml ethanol 99%, containing 0.01 mg/ml TBA to each test tube. Then, all samples were transferred into an ultrasonic bath for 15 min and horizontally shaken for 5 min (100 turns). Subsequently, 1.0 ml of the obtained solution was transferred into glass vials, sealed and subjected to chromatographic analysis.
3
Compressed Ligand Screening on PDAC Organoids
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To perform compressed ligand screening on PDAC organoids, cells were expanded as described, resuspended in OWRNA media with 10% v/v Matrigel, and seeded at 20,000 single cells in 35 µL / well into a flat bottom ultra-low attachment 96-well assay plate (Corning #3474) one day prior to dispensing ligands. A compressed screen was designed by randomly assigning ligands to 68 ligands (Table S2) to 72 pools such that each pool contained 4 or 5 ligands (average of 4.75 ligands per pool). Two such plates were designed, with distinct random assignment of ligands to pools in each plate. Ligands were first dispensed into 25 µL / well of OWRNA media in a 384-well format transfer plate (quadrant-wise). After ligand dispense, each well was backfilled with an additional 50 µL / well of OWRNA media. 65 µL / well was then transferred with a Tecan Freedom Evo 150 from each transfer plate quadrant into the 96-well assay plate with PDAC organoids, making a final total volume of 100 µL / well in the assay plate. Ligand concentration was modified to account for the multi-stage transfer such that final assay plate concentrations are as reported (see Supplemental Table S2). Assay plates were cultured for 7 days at 37C/5% CO2 and processed for single-cell RNA-seq with cell hashing as follows.
4
Standardized RNA Extraction and Analysis
5
Automated RNA Extraction and Transcriptomic Analysis
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Venous whole blood was collected in PAXgene tubes (PreAnalytiX, Hombrechtikon, Switzerland) at enrolment in all cohorts, frozen at −20 °C, and shipped to the South African Tuberculosis Vaccine Initiative (SATVI) Cape Town laboratory. For the CTBC study, RNA was manually extracted with the PAXgene blood RNA kit (Qiagen) according to the manufacturer’s instructions, stored at −80 °C, and later used for transcriptomic analysis. For the other cohorts, RNA was extracted using a high-throughput, standardised, and reproducible fully automated procedure on the Freedom EVO 150 robotic platform (Tecan, Männedorf, Switzerland) with the Maxwell SimplyRNA kit (Promega, Madison, WI, USA). One aliquot of RNA was used immediately for cDNA synthesis and measurement of the RISK11 signature, and a second aliquot was stored at −80 °C, and later used to measure the panel of parsimonious transcriptomic signatures.
6
Dilution Series for qPCR Optimization
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This data set created a dilution series consisting of four 10-fold serial dilution points from 15,000 to 15 molecules, using 10 ng / µl yeast tRNA as a carrier (Roche)
and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8µl samples containing) 0.4µl forward and 0.4µl reverse primer (5µM each), 0.2µl nuclease-free water, 4µl iQ SYBR Green Supermix (Bio-Rad) and 3µl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [9] . This dataset will be referred to as '94replicates-4-dilutions set'. Since our system has 96 reaction channels, we select 44 curve data with concentration gradient from data set for analysis.
and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8µl samples containing) 0.4µl forward and 0.4µl reverse primer (5µM each), 0.2µl nuclease-free water, 4µl iQ SYBR Green Supermix (Bio-Rad) and 3µl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [9] . This dataset will be referred to as '94replicates-4-dilutions set'. Since our system has 96 reaction channels, we select 44 curve data with concentration gradient from data set for analysis.
7
PRRSV Viral RNA Detection and Genotyping
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All 21 samples of group 1 were analysed by real-time RT-PCR for the presence of PRRSV viral RNA. Positive samples were sequenced. Within group 2, all serum samples of the boar stud and the Southeast Asian farm, as well as several tissue samples from the Russian farm, were analysed by PCR. Pigs from group 3 were from farms with continuous PRRSV monitoring by PCR and ELISA with no positive results. The 104 fatteners of group 2 and the samples of groups 3–5 were not tested with PCR in this study.
RNA extraction was performed using the Freedom EVO® 150 (Tecan, Grödig, Austria) automated platform and the Nucleospin® 96 Virus and the Nucleospin® Virus Core kits (Macherey-Nagel, GenXpress, Wiener Neudorf, Austria) for serum and tissue samples, respectively, following the instructions of the manufacturer. The samples were then analysed by a commercial real-time RT-PCR assay that allows the simultaneous detection and differentiation between PRRSV type 1 (EU) and type 2 (NA) genotypes (Life Technologies, Brunn am Gebirge, Austria) on the ABI 7500 Fast Real-Time PCR System (Life Technologies).
RNA extraction was performed using the Freedom EVO® 150 (Tecan, Grödig, Austria) automated platform and the Nucleospin® 96 Virus and the Nucleospin® Virus Core kits (Macherey-Nagel, GenXpress, Wiener Neudorf, Austria) for serum and tissue samples, respectively, following the instructions of the manufacturer. The samples were then analysed by a commercial real-time RT-PCR assay that allows the simultaneous detection and differentiation between PRRSV type 1 (EU) and type 2 (NA) genotypes (Life Technologies, Brunn am Gebirge, Austria) on the ABI 7500 Fast Real-Time PCR System (Life Technologies).
8
PEG-8000 Titration Assay for Protein Stability
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A stock solution of 40% (w/v) PEG-8000 in succinate-Arg buffer was prepared as the titrant. Stock protein solutions were prepared at 9.8 mg/mL in the same buffer. Samples were prepared in triplicate and loaded into a 96–well, clear, flat-bottomed plate (Greiner Bio-One, cat. 655801) using a liquid handling system (Freedom-EVO150, Tecan, Männedorf, Germany) as follows: (i) succinate-Arg buffer was dispensed into each well with volume of 100–180 μL (ii) 20 μL of protein solution was dispensed into each well to aa final concentration of 1 mg/mL; (iii) 0–80 μL of the PEG-8000 stock solution was titrated into each well to give a series of PEG-8000 concentrations from 0–16%; (iv) samples were mixed by slowly aspirating and dispensing the well contents five times (total sample volume in each well was 200 μL). Plates were examined for air bubbles and nephelometry measurements were made immediately using a NEPHELOstar Plus (BMG Labtech, Ortenberg, Germany).
9
High-throughput Candida Growth Profiling
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Overnight cultures of freshly revitalized C. albicans strains and Candida spp. were normalized to a final OD600 of 2 and washed once with PBS. A Freedom EVO 150 liquid-handling robot (Tecan) performed serial dilutions of the cultures and spotted 3 μl of each serial dilution onto YPD agar omnitrays representing one of a wide range of growth conditions listed in Supplementary Table 5 . Each condition was tested a minimum of three independent times and growth data was acquired by a desktop scanner and analyzed using a custom R script for automated spot detection and intensity measurements followed by non-linear curve fitting across the range of serial dilutions and determination of relative growth scores as explained in Supplemental Methods.
10
Comprehensive Immunophenotyping of Leukocytes
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Ten flow cytometry panels of eight-colors each were developed in order to enumerate and phenotype the major circulating leukocyte populations of the 1,000 Milieu Interieur donors. Premix of antibodies were manually made and staining protocol was performed using the Freedom Evo 150 liquid handling system (Tecan). Samples were acquired using MACSQuant analyzers. Panel antibodies and gating strategies were done as previously reported (Hasan et al., 2015 (link)). Converted FCS format files of 313 immunophenotypes were analyzed using FlowJo software version 9.5.3, from which a total of 166 flow cytometry measures were retained including 87 MFI, 76 cell counts and 3 cell ratios (Patin et al., 2018 (link)).
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