The S. pombe QFA was developed from S. cerevisiae QFA as previously described [19 (link), 20 ]. Drug concentrations in media varied depending on whether cells were grown on solid or in liquid media. We observed that 96 well liquid cultures with usual drug concentrations (100 μg/ml G418; 300 μg/ml Hygromycin; 100 μg/ml Nat) grew poorly. Therefore double mutants were cultured in 96-well plates with each well containing 200μl YE5S liquid media with 6.25 μg/ml each (for G418, clonNAT), and 18.75 μg/ml (for Hyg) as final drug concentration were used (G418+Nat for taz1Δ QFA or G418+Hyg for pot1-1 QFA). Liquid cultures were incubated at 30°C for two days without shaking and 384-format robotic spot tests were performed using a Biomek FX robot (Beckman Coulter (UK) Limited, High Wycombe, UK) equipped with a pintool magnetic mount and a 96-pin (2 mm diameter) pintool (V&P Scientific, Inc., San Diego, CA, USA). Photography, image analysis and modelling of fitness were performed as described previously [19 (link)]. Strip charts were generated using the stripchart function from the statistical programming software R [72 ].
Biomek fx robot
Manufactured by Beckman Coulter
Sourced in United States, United Kingdom
The Biomek FX robot is a liquid handling automation system designed for a variety of laboratory applications. It features programmable pipetting capabilities and can be configured with different accessories to handle various sample types and volumes. The Biomek FX robot is intended to automate repetitive liquid handling tasks, improving efficiency and precision in the laboratory.
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Lab products found in correlation
Ribo zero gold, by Illumina (2 mentions)
Bioanalyzer 2100 platform, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Etoposide, by Cayman Chemical (2 mentions)
Staurosporine, by Cayman Chemical (2 mentions)
Ionomycin, by Cayman Chemical (2 mentions)
Tunicamycin, by Cayman Chemical (2 mentions)
Abt 263, by Selleck Chemicals (2 mentions)
Tert butyl hydroperoxide tbhp, by Merck Group (2 mentions)
Architect ci8200 analyser, by Abbott (2 mentions)
Nanodrop spectrophotometer, by Beckman Coulter (2 mentions)
Multimacs mrna isolation kit, by Miltenyi Biotec (1 mentions)
Superscript cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Superscript cdna synthesis, by Thermo Fisher Scientific (1 mentions)
Uracil n glycosylase, by Thermo Fisher Scientific (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
E210 sonication, by Covaris (1 mentions)
19 protocols using biomek fx robot
1
Quantitative Fitness Assay in Fission Yeast
2
Strand-Specific RNA Sequencing Protocol
3
High-Throughput Compound Screening
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Prior to screening, we prepared serial dilutions of the LOPAC compound library (Sigma) in RPMI medium (Life Technologies) to provide a final stock concentration of 50 μM. Taxol/paclitaxel, vinblastine, and U0126, as well as DMSO (all from Sigma) were included as additional spike‐in controls present on all plates. A list of all compounds included in this library is provided with the R/Bioconductor package PGPC and Table EV1 . We seeded 1,250 cells in 45 μl McCoy's medium into each well of 384‐well clear‐bottom microscopy plates (BD Biosciences) and incubated for 1 day at 37°C. 5 μl of compound solution was added using a Beckman Biomek FX robot with 384‐well tip head to yield a final concentration of 5 μM and 0.1% DMSO. Cells were cultured for 2 days at 37°C before analysis. For screening, a single drug concentration of 5 μM was used.
4
RNA-Seq Profiling of Murine Cell Lines
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Total RNA was extracted from cells from six mice (3× K, 3× KM) using the RNeasy mini kit (Qiagen). RNA quality control was performed using the 2100 Bioanalyzer platform (Agilent). A total of 500 ng total RNA was used as a starting material for the RNA sequencing libraries, which were prepared using the TruSeq strand-unspecific protocol with Ribo-Zero Gold (Illumina) and sequenced on the Illumina HiSeq 2000 platform with 2 × 51 cycles according to the manufacturer's instructions. In order to minimize batch effects, samples were processed on a Beckman Biomek FX robot following the manufacturer's instructions.
5
Octanol-Water Partition Coefficient Determination
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The
method for measurement
of log D was based on the traditional shake
flask technique, using UPLC with quantitative mass spectrometry (MS)
to measure the relative octanol and aqueous concentrations. 1-Octanol
(HPLC grade, ≥99%, Sigma-Aldrich) and a 10 mM phosphate buffer
[Na2HPO4·2H2O (p.a. grade, Merck)
and NaH2PO4·H2O (p.a. grade,
Merck)] were used. Equal parts of buffer and 1-octanol were vigorously
mixed in a separation funnel three times (at least 15 min between
each mixing) to saturate the solutions. The mixture was left overnight
to separate the upper octanol phase from the lower buffer phase before
being used in the experiments. Compounds were assayed in pools of
eight, and four dilutions of buffer and octanol samples were analyzed
and evaluated for log D calculation. All liquid
transfers were performed with a Beckman Biomek FX robot, and samples
were analyzed on a fast-scanning triple quadropole mass spectrometer
(Waters Micromass TQD with MassLynx 4.1) coupled to a Waters Acquity
Ultra Performance LC using a Acquity UPLC HSS T3 1.8 μm, 2.1
mm × 50 mm or Acquity UPLC BEH C18 1.7 μm, 2.1 mm ×
50 mm column.
method for measurement
of log D was based on the traditional shake
flask technique, using UPLC with quantitative mass spectrometry (MS)
to measure the relative octanol and aqueous concentrations. 1-Octanol
(HPLC grade, ≥99%, Sigma-Aldrich) and a 10 mM phosphate buffer
[Na2HPO4·2H2O (p.a. grade, Merck)
and NaH2PO4·H2O (p.a. grade,
Merck)] were used. Equal parts of buffer and 1-octanol were vigorously
mixed in a separation funnel three times (at least 15 min between
each mixing) to saturate the solutions. The mixture was left overnight
to separate the upper octanol phase from the lower buffer phase before
being used in the experiments. Compounds were assayed in pools of
eight, and four dilutions of buffer and octanol samples were analyzed
and evaluated for log D calculation. All liquid
transfers were performed with a Beckman Biomek FX robot, and samples
were analyzed on a fast-scanning triple quadropole mass spectrometer
(Waters Micromass TQD with MassLynx 4.1) coupled to a Waters Acquity
Ultra Performance LC using a Acquity UPLC HSS T3 1.8 μm, 2.1
mm × 50 mm or Acquity UPLC BEH C18 1.7 μm, 2.1 mm ×
50 mm column.
6
Quantifying Cell Death in C2C12 Cells
7
Automated High-Throughput Genomic DNA Extraction
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Total genomic DNA was extracted from c. 1.5 cm2 frozen leaf material using the DNeasy 96 Plant Kit (Qiagen, Hilden, Germany) following modified manufacturer’s instructions. Briefly, leaf material was ground to a powder using a MM300 Mixer Mill (Retsch, Haan, Germany), to which the AP1 lysis buffer containing RNAse A and Reagent DX was added and homogenised with the Mixer Mill. Buffer AP2 was added to the homogenised leaf/AP1 mixture and incubated at −20 °C for 10 min. Samples were then centrifuged at 5800 g for 5 min. For a high-throughput, automated approach, the remainder of the extraction protocol was carried out using a Biomek FX robot (Beckman-Coulter, Brea, CA, USA) with a customised program, with the extraction being performed following the manufacturer’s instructions. DNA quality was assessed using a 1% (w/v) agarose gel in 0.5 × TBE with DNA ladder, Hyperladder I (Bioline, London, UK).
8
Competitive Counterflow Assay for OATP Transporters
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For the competitive counterflow assay, OATP1B1- and OATP1B3-expressing cells were preloaded on 96-well plates at room temperature for 2 h with 100 μl of 10 nM [3H]-estradiol-17β-glucuronide in uptake buffer (116.4 mM NaCl, 5.3 mM KCl, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.5 mM D-glucose and 20 mM HEPES, pH adjusted to 7.4 with Tris). (Gui et al., 2008 (link)). Then efflux was initiated by the addition of 1 µl of a stock solution of 100 μM, 1 mM, or 10 mM test compound dissolved in DMSO, or DMSO for the control condition (final DMSO concentration was 1%) using a Biomek FX robot (Beckman Coulter Inc., Brea, CA). After 30 min, efflux was stopped by washing the wells three times with 100 µl of room temperature uptake solution with a Wellwash™ Versa microplate washer (Thermo Fisher Scientific Inc., Waltham, MA); cells were solubilized with 150 μl of Triton X-100. One hundred microliters of this solution were transferred to 96-well Flexible PET Microplates (PerkinElmer). After adding 200 μl of Optiphase HiSafe, samples were counted using a MicroBeta microplate scintillation counter (PerkinElmer).
9
Barcoding Cells for Single-Cell Analysis
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All pipetting steps were implemented with a Biomek Fx Robot (Beckmann Coulter).
Remove PBS from washing by sucking all liquid at a height of ca 2 mm from well bottom from the middle of the well using a gentle flow rate (estimated residual volume ca 30 µl).
Pre‐dilute 4 ul barcoding solution with 65 µl of PBS and add to each well.
Incubate plates for 1 h shaking at 200 RPM.
Wash plates four times with 150 µl of 1x Cell Staining Medium (CSM, PBS (pH 7.4, Gibco) 0.5% bovine serum albumin (Sigma)).
10
Screening Yeast Double Mutants
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Double mutants obtained from SGA were inoculated into liquid (final media: SC-ARG, −HIS, −LYS, −LEU, +canavanine, +thialysine, +geneticin, +hygromycin, and +nourseothricin) at 20° in 96-wells plates and grown to saturation. Cultures were spotted on solid agar-plates in 384-format using a Biomek FX robot [Beckman Coulter (UK) Limited, High Wycombe, UK], and growth assays carried out as in Addinall et al. (2011) (link), except we used concentrated cultures, without diluting in water, for lyp1 Δ (control), pol1 -4, pol2 -12, and cdc2 -2 experiments. Plates were imaged automatically, usually every 4 hr. For each genotype, we performed four independent genetic crosses and analyzed two growth curves from each cross (eight growth curves in total). In some cases, however, we examined more than eight cultures (i.e., up to 144 for his3 Δ, repeated across several plates) and for others less than eight (i.e., down to four because of to technical errors). The specific number of growth curves analyzed for each double mutant can be found in File S1 .
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