Rotor pinning robot

Manufactured by Singer Instruments

Sourced in United Kingdom

The RoToR pinning robot is a laboratory instrument designed for automated pinning of microbial samples onto agar plates. The device precisely positions and applies samples onto the agar surface, enabling efficient and consistent inoculation for various microbiological applications.

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Lab products found in correlation

Facscanto ruo hts flow cytometer, by BD (3 mentions) Lsrfortessa with high throughput sampler, by BD (1 mentions) Sanger sequencing, by Beckman Coulter (1 mentions)

Spelling variants of this product

ROTOR robot (11 citations) ROTOR HDA pinning robot (4 citations) Singer RoToR HDA pinning robot (2 citations)

6 protocols using rotor pinning robot

High-Throughput Mutant Archiving and Screening

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We split biological replicates for each transcription mutation or
gene deletion into two sets (Array #1 and Array #2). We then arrayed the
mutants within each set in triplicate with randomized positions in a
32×48 array of 1536 colonies. To minimize edge effects (French et al., 2016 (link)), we filled the
outermost two columns and rows of the 1536-colony array with wild-type
controls and only analyzed the inner positions. Mutants were split according
to antibiotic resistance phenotype (Cam^r and Kan^r)
into 16 groups that corresponded to each of the 16 96-well plates that would
comprise the 1536 array. Based on the final position in the 1536-well array,
spaces in each 96-well plate were devoted to wild-type (either BW25113 or
BW25113 rpoBC-cat) and used as “dummy”
colonies that would grow in all conditions.
For storage, plates were grown overnight at 37 °C with
shaking at 900 rpm in a humidified platform shaker (Infors HT). Glycerol was
added to a final concentration of 12.5%, and aliquots of each plate were
stored at −80 °C in a 96-well format. The two 1536-colony
arrays were assembled by thawing copies of the 2×16 96-well plates
and using a Rotor pinning robot (Singer Instruments) to spot the plates,
first into 2×4 384-colony plates, and finally into two 1536-colony
format plates (Array #1 and Array #2).

Shiver A.L., Osadnik H., Peters J.M., Mooney R.A., Wu P.I., Henry K.K., Braberg H., Krogan N.J., Hu J.C., Landick R., Huang K.C, & Gross C.A. (2021). Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs. Molecular cell, 81(10), 2201-2215.e9.

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Genome-Wide SUS-GFP Screening in Yeast

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The SUS-GFP query strain was crossed with the SPOCK collection, consisting of 5,808 yeast non-essential null and essential DaMP gene mutants (Jaeger, Ornelas et al., 2017 ). All library manipulations including selection of diploids, sporulation, and selection of haploids were done using standard EMAP technology (Collins et al., 2010 (link)) using a RoToR pinning robot (Singer Instruments, Taunton, UK) and Uracil and G418 selection. Once SUS-GFP was introduced into every yeast viable null and hypomorphic allele, the yeast array was grown for 24 hrs at 30°C. The resulting array was then transferred to liquid YPD media (50 μL/well) using the RoToR. After further incubation at 30°C for 2 days, we analyzed SUS-GFP stabilization with an LSR Fortessa with High Throughput Sampler (BD Biosciences, San Jose) set to collect 10,000 events per sample. GFP and autofluorescence were excited at 488 nm and 405 nm respectively and detected with 510/25 and 450/50 bandpass filters. Data was analyzed in FlowJo version 9. GFP+ events were gated by plotting autofluorescence versus GFP fluorescence.

Neal S., Jaeger P.A., Duttke S., Benner C., Glass C., Ideker T, & Hampton R. (2018). The Dfm1 derlin is required for ERAD retrotranslocation of integral membrane proteins. Molecular cell, 69(2), 306-320.e4.

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High-Throughput Yeast Fluorescence Assay

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Yeast cells were inoculated into 200 μl SC medium and grown to saturation in 96-well plates. The cultures were then diluted into fresh medium by pinning to a new 96-well plate using a RoToR pinning robot (Singer Instruments) and incubated at 23°C for 20–24 h to 1 × 10⁶–8 × 10⁶ cells/ml. Flow cytometry was performed on a FACSCanto RUO HTS flow cytometer (BD Biosciences) equipped with a high-throughput sample loader, a 561 nm laser with 600 nm long pass and 610/20 nm band pass filters for mCherry, and a 488 nm laser with 505 nm long pass and 530/30 nm band pass filters for sfGFP. Data analysis was performed in R (R Core Team, 2016) with the flowCore and flowWorkspace packages using a custom script. Briefly, the events were gated for single cells using forward and side scatter pulse width, followed by gating for fluorescent cells. The median intensity of a negative control was subtracted from each cell. The median sfGFP intensity of each sample was used for further analysis. Unless otherwise stated, each experiment was performed using two biological replicates with three technical replicates each.

Kats I., Reinbold C., Kschonsak M., Khmelinskii A., Armbruster L., Ruppert T, & Knop M. (2021). Up-regulation of ubiquitin–proteasome activity upon loss of NatA-dependent N-terminal acetylation. Life Science Alliance, 5(2), e202000730.

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Yeast Strain Construction and Plasmid Validation

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The yeast strains used in this study are listed in Supplemental Material, Table S2. Strains were constructed using standard techniques, and standard yeast growth medium including 2% (w/v) of the indicated carbon source (Sherman 2002 (link)). Yeast plasmids were created using the gap-repair cloning technique, which combines a linearized plasmid with PCR products using in vivo recombination. All PCR products were generated using primers from Sigma Life Science and PfuII Ultra proof reading polymerase (Agilent Technologies, UK). All plasmid constructs (listed in Table S3) were validated using Sanger sequencing (Beckman Coulter Genomics, UK). Selective ploidy ablation (SPA) screening followed the established protocol using the donor strain W8164-2B (Reid et al. 2011 (link)), and utilizing a ROTOR pinning robot (Singer Instruments, UK).

Ólafsson G, & Thorpe P.H. (2016). Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions. G3: Genes|Genomes|Genetics, 6(8), 2531-2542.

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Flow Cytometry-based Protein Regulation Assay

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Yeast strains containing the desired plasmids were inoculated into 200 µl SC medium lacking the appropriate amino acids for plasmid selection and grown to saturation in 96-well plates. The cultures were then diluted into fresh medium by pinning to a new 96-well plate using a RoToR pinning robot (Singer Instruments) and incubated at 23°C for 20-24 h to 1x10 6 -8x10 6 cells/ml. Flow cytometry was performed on a FACSCanto RUO HTS flow cytometer (BD Biosciences) equipped with a high-throughput sample loader, a 561 nm laser with 600 nm long pass and 610/20 nm band pass filters for mCherry, and a 488 nm laser with 505 nm long pass and 530/30 nm band pass filters for sfGFP. Data analysis was performed in R (R Core Team, 2016) with the flowCore and flowWorkspace packages using a custom script. Briefly, the events were gated for mCherry-and sfGFP-positive cells, the median intensity of a negative control was subtracted from each channel, and the mCherry/sfGFP ratio was calculated for each cell. The median mCherry/sfGFP ratio of each sample was used for further analysis. Unless otherwise stated, each experiment was performed using two biological replicates with three technical replicates each. To account for growth rate differences, sample mCherry/sfGFP ratios were normalized to the stable Ubi-TH-eK-tFT reporter (plasmid pAnB19-TH, Table S2), which was measured in each strain background.

Kats I., Kschonsak M., Khmelinskii A., Armbruster L., Ruppert T., & Knop M. (2020). Mechanisms of up-regulation of Ubiquitin-Proteasome activity in the absence of NatA dependent N-terminal acetylation.

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Flow Cytometry Quantification of Fluorescent Proteins

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Yeast cells were inoculated into 200 µl SC medium and grown to saturation in 96-well plates. The cultures were then diluted into fresh medium by pinning to a new 96-well plate using a RoToR pinning robot (Singer Instruments) and incubated at 23°C for 20-24 h to 1x10 6 -8x10 6 cells/ml. Flow cytometry was performed on a FACSCanto RUO HTS flow cytometer (BD Biosciences) equipped with a high-throughput sample loader, a 561 nm laser with 600 nm long pass and 610/20 nm band pass filters for mCherry, and a 488 nm laser with 505 nm long pass and 530/30 nm band pass filters for sfGFP. Data analysis was performed in R (R Core Team, 2016) with the flowCore and flowWorkspace packages using a custom script. Briefly, the events were gated for single cells using forward and side scatter pulse width, followed by gating for fluorescent cells. The median intensity of a negative control was subtracted from each cell. The median sfGFP intensity of each sample was used for further analysis. Unless otherwise stated, each experiment was performed using two biological replicates with three technical replicates each.

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