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96 well deep well plate

Manufactured by Corning
Sourced in United States

The 96-well deep well plate is a laboratory equipment designed for high-throughput sample preparation and storage. It features a standard 96-well format with increased well depth, allowing for larger sample volumes compared to traditional microplates. The plate is typically made of polypropylene or other chemically resistant materials, ensuring compatibility with a wide range of laboratory applications.

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6 protocols using 96 well deep well plate

1

Arabinose-Induced Luminescence Assay

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S536 and S1367 cells were transformed with APs and CPs as indicated in Supplementary table 2. Freshly saturated cultures of single colonies grown in Davis Rich Media19 (link) (DRM) plus maintenance antibiotics were diluted 500-fold into DRM media with maintenance antibiotics in a 96-well deep-well plate (Axygen) and induced with indicated concentrations of arabinose (Gold Biotechnology) before incubation for 2 h at 37 °C with shaking at 230 RPM. 150 μL of cells per well were then transferred to a 96-well black-walled clear-bottomed plate with a transparent lid (Costar). 600 nm absorbance and luminescence were read at 15-min intervals over an 8-h kinetic cycle with shaking at 230 RPM between reads using a Tecan Spark multimode microplate reader (Tecan). Single read data were taken at peak luminescence value (4–5 h post-induction). OD600-normalized luminescence values were determined by dividing raw luminescence by background-subtracted (DRM only) 600 nm absorbance.
For phage-induced luciferase timecourse assay, S536 and S2060 cells were transformed with APs and diluted in DRM as described above. Cells were grown to an OD600 of 0.4 and were inoculated with selection phage at an initial titer of 5 × 104 pfu/mL. 150 μL of cells per well were immediately transferred to a plate for luminescence and optical density reading in a kinetic cycle as described above.
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2

Inducible Protein Expression in S2060 Cells

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S2060 cells were transformed with the AP(s) and CP(s) of interest as described above. Overnight cultures of single colonies grown in 2xYT media supplemented with maintenance antibiotics were diluted 500-fold into DRM media17 (link) with maintenance antibiotics in a 96-well deep well plate (Axygen). The plate was sealed with a porous sealing film and grown at 37˚C with shaking at 230 RPM for 4 h, whereupon the culture reached OD600 ~ 0.4–0.6. The cells were then induced with 100 ng/mL anhydrotetracycline (aTc; Fluka) and 5 μM arabinose (Gold Biotechnology) before incubation for an additional 1 h at 37˚C with shaking at 230 RPM. 120 μL of cells was transferred to a 96-well black-walled clear-bottomed plate (Costar), then 600 nm absorbance and luminescence were read using an Infinite M1000 Pro microplate reader (Tecan). OD600-normalized luminescence values were obtained by dividing raw luminescence by background-subtracted 600 nm absorbance. The background value was set to the 600 nm absorbance of wells containing DRM only.
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3

Inducible Protein Expression in S2060 Cells

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S2060 cells were transformed with the AP(s) and CP(s) of interest as described above. Overnight cultures of single colonies grown in 2xYT media supplemented with maintenance antibiotics were diluted 500-fold into DRM media17 (link) with maintenance antibiotics in a 96-well deep well plate (Axygen). The plate was sealed with a porous sealing film and grown at 37˚C with shaking at 230 RPM for 4 h, whereupon the culture reached OD600 ~ 0.4–0.6. The cells were then induced with 100 ng/mL anhydrotetracycline (aTc; Fluka) and 5 μM arabinose (Gold Biotechnology) before incubation for an additional 1 h at 37˚C with shaking at 230 RPM. 120 μL of cells was transferred to a 96-well black-walled clear-bottomed plate (Costar), then 600 nm absorbance and luminescence were read using an Infinite M1000 Pro microplate reader (Tecan). OD600-normalized luminescence values were obtained by dividing raw luminescence by background-subtracted 600 nm absorbance. The background value was set to the 600 nm absorbance of wells containing DRM only.
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4

Standardized Bacterial DNA Extraction

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DNA extraction was carried out in batches of 84 samples. Paired mother, child, and researcher samples were processed in the same batch. To minimize differences in isolation between batches of the same kit and daily variation, kit blanks and blank eNAT solution and swabs were included in each batch. The tubes were thawed and thoroughly homogenized. From each tube 400 microliter eNAT solution was transferred to an assigned well in a 96 well deep well plate (Axygen scientific Inc., CA, USA). Subsequently, the swab from the same tube was cut off with sterile scissors and transferred to the same well, followed by the addition of 250 microliter of 0.1 mm Zyrconia beads. The deep well plate was sealed with a silicone lid and placed in a Mini-BeadBeater-96 (BioSpec Products, Bartlesville, OK, USA). The plate was subjected to bead beating for four series of 2 min at 2100 oscillations/ min. The bacterial DNA concentration in the samples (after DNA purification) was determined by qPCR, with universal primers specific to the bacterial 16S rRNA gene [16] .
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5

Oropharyngeal Swab Collection and RNA Extraction

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Oropharyngeal (OP, Improve Medical, China) swabs were used to collect OP/mid-turbinate samples from individuals at walk-up testing centers in Berkeley, CA. Swabs were transported to the IGI Clinical Laboratory in 4mL of 2x DNA/RNA Shield (Zymo Research, Irvine CA), diluted to 1x with sterile phosphate buffered saline, pH 7.4 (Gibco, Gaithersburg, MD). Samples were kept at 4°C until 450μL was aliquoted into 96-well deep-well plates (Axygen, San Francisco CA) with the Microlab STARlet liquid-handling robot (Hamilton, Reno NV). RNA extraction was performed on the Vantage liquid handling system (Hamilton) with the MagMax Viral/Pathogen Nucleic Acid Isolation Kit (Applied Biosystems), according to the manufacturer’s instructions, with the exception of omitting MS2 spike-in for the IGI-LuNER assay. Final RNA elution volume was 22μL. RNA was stored at −80°C or arrayed into 384-well master mix plates on the Vantage liquid-handling system (5μL of sample into 7.5μL master mix) for RT-qPCR.
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6

Standardized COVID-19 Sample Processing

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Oropharyngeal (OP, Improve Medical, China) swabs were used to collect OP/mid-turbinate samples from individuals at walk-up testing centers in Berkeley, CA. Swabs were transported to the IGI Clinical Laboratory in 2mL of 2x DNA/RNA Shield (Zymo Research, Irvine CA), diluted to 1x with sterile phosphate buffered saline, pH 7.4 (Gibco, Gaithersburg, MD). Samples were kept at 4°C until 450μL (single extractions) or 112.5μL (fourplex pooled extractions) was aliquoted into 96-well deep-well plates (Axygen, San Francisco CA) with the Microlab STARlet liquid-handling robot (Hamilton, Reno NV). RNA extraction was performed on the Vantage liquid handling system (Hamilton) with the MagMax Viral/Pathogen Nucleic Acid Isolation Kit (Applied Biosystems), according to the manufacturer’s instructions, with the exception of omitting MS2 spike-in for the LuNER assay. Final RNA elution volume was 22μL. RNA was stored at -80°C or immediately arrayed into 384-well master mix plates on the Vantage liquid-handling system (5μL of sample into 7.5μL master mix) for RT-qPCR.
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