Ez rdm

Manufactured by Teknova

Sourced in United States

The EZ-RDM is a rapid dispenser for microbiological media. It efficiently and accurately dispenses media into Petri dishes, tubes, and other containers. The device is designed for consistent and repeatable dispensing, ensuring standardized sample preparation for microbiological analyses.

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Lab products found in correlation

Bacto agar, by BD (2 mentions) Macsquant vyb, by Miltenyi Biotec (2 mentions) E coli dh5α cells, by Thermo Fisher Scientific (2 mentions) Synergy htx plate reader, by Agilent Technologies (1 mentions) Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions) Minispin, by Eppendorf (1 mentions) Micropulser, by Bio-Rad (1 mentions) Macsquantify 2, by Miltenyi Biotec (1 mentions) Macsquant vyb flow cytometer, by Miltenyi Biotec (1 mentions) Gen5 2, by Agilent Technologies (1 mentions) Synergy htx, by Agilent Technologies (1 mentions)

12 protocols using ez rdm

Monitoring Bacterial Growth and Fluorescence

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Single colonies from LB plates were inoculated in 400 μl of EZ-RDM (Teknova) supplemented with the appropriate antibiotics and grown in 96-deep-well plates at 37°C with shaking overnight 900 rpm on a Heidolph titramax 1000. 100 μl of the overnight culture were transferred into flat, clear-bottomed black 96-well plates (Corning) and the OD₆₀₀ and fluorescence were measured in a Biotek Synergy HTX plate reader. For mRFP1 (referred to as mRFP) detection, the excitation wavelength was 540 nm and emission wavelength was 600 nm. For sfGFP detection, the excitation wavelength was 485 nm and emission wavelength was 528 nm. For mTagBFP2 (referred to as mBFP) detection, the excitation wavelength was 400 nm and emission wavelength was 450 nm. At least three biological replicates were used for all experiments unless stated otherwise.
For kinetic experiments, overnight cultures in stationary phase were subcultured to OD₆₀₀ 0.1 in 200 μl of EZ-RDM (Teknova) supplemented with the appropriate antibiotics. Cultures were grown in flat, clear-bottomed black 96-well plates (Corning) at 37°C in a Biotek Synergy HTX plate reader set to shake at 1200 RPM. Surrounding wells were filled with 200 μl of water to maintain humidity. The OD₆₀₀ and fluorescence were measured every 30 min for 16 h.

Cardiff R.A., Faulkner I.D., Beall J.G., Carothers J.M, & Zalatan J.G. (2024). CRISPR-Cas tools for simultaneous transcription & translation control in bacteria. Nucleic Acids Research, 52(9), 5406-5419.

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Single-Molecule Tracking of Penicillin-Binding Proteins

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Cells expressing fluorescently labelled proteins were grown to saturation overnight in the rich medium EZ-RDM (Teknova, Hollister, CA, USA)44 (link) with 0.2% glucose and then diluted 1:100 in fresh medium and incubated with shaking at 30 °C for 3.5 h. Cells were spotted onto 1% agarose pads with EZ-RDM+0.2% glucose and covered with argon plasma-cleaned coverslips. For drift correction, phase-contrast images were taken interlaced with fluorescence images. To capture PBP1A/1B/2 dynamics, image acquisition was alternated between: 1) an exposure with 561-nm laser at ∼1 kW cm⁻² to image single PAmCherry molecules and 2) a simultaneous exposure with a brightfield light-emitting diode and 405-nm laser (∼0.50 kW cm⁻² for PBP1A and PBP2, ∼0.05 kW cm⁻² for PBP1B) to capture phase-contrast images and to photoactivate PAmCherry molecules. In single-particle tracking experiments under antibiotic treatment, agarose pads were cast with the appropriate antibiotic (cefsulodin, mecillinam, ampicillin, and cefmetazole at 100 μg ml⁻¹) and cells were directly spotted from liquid culture.

Lee T.K., Meng K., Shi H, & Huang K.C. (2016). Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells. Nature Communications, 7, 13170.

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Genomic CRISPR Interference and Cleavage

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The in vivo CRISPRi^{28 (link)} and cleavage assays described below utilize E. coli MG1655 containing genomically-integrated and constitutively expressed Green fluorescent protein (GFP) and Red fluorescent protein (RFP). Standard cloning techniques were used to create all plasmids. Plasmid construction and retention was ensured with AmpR and CmR as selectable markers¹⁵. EZ-rich defined growth media (EZ-RDM, Teknova) was used in all CRISPRi assay fluorescent measurements. 2xYT (LB) with the addition of 1.5% Bacto Agar (BD) was used for all plating assays.

Liu J.J., Orlova N., Oakes B.L., Ma E., Spinner H.B., Baney K.L., Chuck J., Tan D., Knott G.J., Harrington L.B., Al-Shayeb B., Wagner A., Brötzmann J., Staahl B.T., Talyor K.L., Desmarais J., Nogales E, & Doudna J.A. (2019). CRISPR-CasX is an RNA-dominated enzyme active for human genome editing. Nature, 566(7743), 218-223.

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Single-cell flow cytometry of bacterial cultures

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Cells were inoculated in EZ-RDM (Teknova) supplemented with appropriate antibiotics and grown in 96-deep-well plates at 37 °C, 220 RPM overnight. Late stationary phase cultures were then diluted 1:40 in PBS and analyzed on a MACSQuant VYB flow cytometer (Miltenyi Biotec). To enrich for single cells, a side scatter threshold trigger (SSC-H) was applied. To gate for single bacterial cells, we first selected events along the diagonal of the SSC-H vs. SSC-A plot^{59 (link)}. We then excluded events that appeared on the edges of the SSC-A vs. FSC-A plot, and events that appeared on the edge of the fluorescence histogram (Supplementary Fig. 9).
For inducible CRISPR system construction with pTet or pBAD promoters, strains were inoculated in 3 mL LB medium supplemented with antibiotics and grown overnight at 37 °C, 220 RPM. On the next day, they were diluted 1:100 in 500 µL EZ-RDM supplemented with antibiotics and induced with 1 µM anhydrotetracycline (aTc) for pTet or 100 mM l-arabinose for pBAD. Non-induced controls were prepared for each strain. For pTet, cells were grown at 37 °C, 220 RPM overnight. For pBAD, cells were grown at 37 °C, 220 RPM for 6 h. In both cases, fluorescence was assayed via flow cytometry as described above.

Dong C., Fontana J., Patel A., Carothers J.M, & Zalatan J.G. (2018). Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria. Nature Communications, 9, 2489.

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Electroporation of E. coli Cells with Labeled tRNA

Cited 1 time

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E. coli DH5α cells (Invitrogen) were previously diluted five times on 10% glycerol (OD₆₀₀ [optical density at 600 nm] = 60 ± 10) and stored in aliquots of 20 µL at −80 °C. One aliquot of cells (SQ171, for experiments with aminoglycoside-resistant ribosomes) was mixed with 1.5 pmol of Phe-[Cy5]tRNA^Phe or 2 pmol of fMet-[Cy5]tRNA^fMet (prepared as in ref. 28 (link)) and incubated 1 min on ice. The mixture was transferred to a cold (∼4 °C) electroporation cuvette (MBP, 1 mm) and pulsed with 1.9 kV with a MicroPulser (Bio-Rad), which generated a decay time constant of 5.8 ± 0.1. Immediately after, 1 mL of EZ RDM (Teknova) + 0.2% glucose was added to the cuvette to gently resuspend the cells. The suspension was pipetted down to a culture tube and incubated for 30 min at 37 °C and shaking at 200 rpm. The cells were pelleted at RCF 2415 × g for 3 min (MiniSpin, Eppendorf) and washed three times with fresh RDM (room temperature) to remove nonelectroporated tRNA. Cells were finally resuspended in RDM (OD₆₀₀ = 0.02) containing 1 µM SYTOX blue dead cell stain (Invitrogen).

Aguirre Rivera J., Larsson J., Volkov I.L., Seefeldt A.C., Sanyal S, & Johansson M. (2021). Real-time measurements of aminoglycoside effects on protein synthesis in live cells. Proceedings of the National Academy of Sciences of the United States of America, 118(9), e2013315118.

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Orthogonal CRISPR-based Regulation System

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For each mechanism, orthogonal cgRNA/trigger pairs were designed
using the reaction pathway engineering tools within NUPACK (nupack.org).^{31 (link),32 (link)} For bacterial studies, a control gRNA or a cgRNA/trigger plasmid
was transformed into a modified E. coli MG1655 strain
expressing genomically incorporated mRFP and sfGFP.^{4 (link)} Strains were grown overnight in EZ-RDM (Teknova) and then
diluted and grown to mid log phase (≈4 h). Cell density was
normalized with fresh medium containing aTc for induction of silencing
dCas9 expression (and IPTG for the bacterial terminator switch experiments
only). Induced cells were grown for 12 h, with end-point fluorescence
measured via flow cytometry. For mammalian studies, a cgRNA expression
plasmid and a trigger expression plasmid were cotransfected with a
plasmid expressing an inducing dCas9-VPR fusion^{33 (link)} and a reporter plasmid containing a gRNA binding site upstream
of a minimal CMV promoter for dTomato expression.^{34 (link),35 (link)} The four plasmids were transiently transfected into HEK 293T cells
with Lipofectamine 3000 and grown for 24 h, with end-point fluorescence
measured via flow cytometry. Data analysis was performed on cells
expressing high levels of both cgRNA and trigger fluorescent protein
transfection controls. No unexpected or unusually high safety hazards
were encountered.

Hanewich-Hollatz M.H., Chen Z., Hochrein L.M., Huang J, & Pierce N.A. (2019). Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology. ACS Central Science, 5(7), 1241-1249.

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Live-cell Imaging and Fixation Protocol

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For live-cell imaging, single colonies were picked from LB plates and cultured overnight in EZ Rich Defined Medium (EZRDM, Teknova) with 0.4% glucose at room temperature (RT) with shaking. The next morning, cells were reinoculated into fresh EZRDM with 0.4% glucose and grown at RT until they reached mid-log phase (OD₆₀₀, 0.3–0.4). For simultaneous visualization of DNA site markers (results are not reported here), cells were collected and resuspended in fresh EZRDM supplemented with 0.3% l-arabinose and 0.4% glycerol and allowed to grow for 2 additional hours; these cells were collected via centrifugation and imaged immediately. For fixed-cell experiments, cells were grown accordingly and fixed in 3.7% (vol/vol) paraformaldehyde (16% paraformaldehyde, EM grade, EMS) for 15 min at RT, washed with 1× PBS and imaged immediately.

Bohrer C.H., Yang X., Thakur S., Weng X., Tenner B., McQuillen R., Ross B., Wooten M., Chen X., Zhang J., Roberts E., Lakadamyali M, & Xiao J. (2021). A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM. Nature methods, 18(6), 669-677.

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Flow Cytometric Analysis of Single Cells

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Single colonies from LB plates were inoculated in 400 μL EZ-RDM
(Teknova) supplemented with appropriate antibiotics and grown in 96-deep-well
plates at 37 °C, 900 RPM on a Heidolph titramax 1000. Cultures were grown
overnight and then diluted in 1:100 in Dulbecco’s phosphate-buffered
saline (PBS) and analyzed on a MACSQuant VYB flow cytometer with the
MACSQuantify 2.8 software (Miltenyi Biotec). To select single cells, we used a
previously-described gating procedure.^{2 (link)} A side scatter threshold trigger (SSC-H) was applied to
select for single cells until 10000 events were collected. FlowJo 10.0.7
software was used to apply a narrow gate along the diagonal line on the SSC-H vs
SSC-A plot to exclude the events where multiple cells were grouped together.
Within the selected population, events that appeared on the edges of the FSC-A
vs. SSC-A plot and the fluorescence histogram were excluded.

Wroe S.F., Kelsey G., Skinner J.A., Bodle D., Ball S.T., Beechey C.V., Peters J, & Williamson C.M. (2000). An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3342-3346.

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Genomic CRISPR Interference and Cleavage

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Single-cell flow cytometry analysis

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Single colonies from LB plates were inoculated in 500 μL EZ-RDM (Teknova) supplemented with appropriate antibiotics and grown in 96-deep-well plates at 37 °C and shaking. Cultures were grown overnight at 37 °C and shaking and then diluted in 1:50 in Dulbecco’s phosphate-buffered saline and analyzed on a MACSQuant VYB flow cytometer with the MACSQuantify 2.8 software (Miltenyi Biotec). A side scatter threshold trigger (SSC-H) was applied to enrich for single cells until 10000 events were collected. The FlowJo 10.0.7 software was used to apply a narrow gate along the diagonal line on the SSC-H vs SSC-A plot was selected to exclude the events where multiple cells were grouped together. Within the selected population, events that appeared on the edges of the FSC-A vs. SSC-A plot and the fluorescence histogram were excluded.

Fontana J., Dong C., Kiattisewee C., Chavali V.P., Tickman B.I., Carothers J.M, & Zalatan J.G. (2020). Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements. Nature Communications, 11, 1618.

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