96 well pcr plate

Manufactured by Eppendorf

Sourced in Germany

The 96-well PCR plate is a laboratory consumable used for performing polymerase chain reaction (PCR) experiments. It provides a standardized format with 96 individual wells, allowing for multiple samples to be processed simultaneously. The plate is designed to be compatible with common PCR thermal cyclers and pipetting devices, facilitating efficient and high-throughput PCR workflows.

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Lab products found in correlation

Qx200 droplet generator, by Bio-Rad (7 mentions) Droplet generation oil, by Bio-Rad (5 mentions) Qx200 droplet reader, by Bio-Rad (5 mentions) Droplet reader, by Bio-Rad (4 mentions) Ddpcr supermix for probe, by Bio-Rad (4 mentions) Quantasoft software, by Bio-Rad (4 mentions) Droplet generation oil for probe, by Bio-Rad (4 mentions) Disposable plastic cartridge, by Bio-Rad (4 mentions) Ddpcr master mix, by Bio-Rad (4 mentions) Droplet generator, by Bio-Rad (3 mentions) T100tm thermal cycler, by Bio-Rad (3 mentions) Dg8 cartridge, by Bio-Rad (3 mentions) Qx100 droplet digital pcr system, by Bio-Rad (3 mentions) C1000 touch thermal cycler, by Bio-Rad (3 mentions) Adhesive pcr plate seals, by Thermo Fisher Scientific (2 mentions) Facsaria 2 cell sorter, by BD (2 mentions) Droplet generator oil, by Bio-Rad (2 mentions) Droplet generator cartridge, by Bio-Rad (2 mentions) Quantasoft analysis software, by Bio-Rad (2 mentions) Px1 pcr plate sealer, by Bio-Rad (2 mentions)

37 protocols using 96 well pcr plate

Chemically Competent E. coli Transformation

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Chemically competent Escherichia coli XL-1 Blue cells were prepared by the Inoue method^{29 , 30} and had a transformation efficiency of at least 10⁷ colonies per 1 µg plasmid pBR322. The assembled fragments were transformed into the competent cells using 20 μL of cell suspension and 2 μL assembly mix for GPCR mutagenesis or 50 μL cell suspension and 5 μL assembly mix for ETR1 mutagenesis. Cells for transformations were thawed on ice, aliquoted, incubated with DNA for 10–20 min, heat shocked for 1 min at 42 °C, incubated on ice for 2 min and plated on LB agar plates with 150 μg mL⁻¹ ampicillin. A quarter of LB agar plate was used for each V2R mutant, while the transformed cells of other constructs were spread over a whole plate. Plated cells were incubated at 37 °C for 16–24 h.
PCR reactions, heat shock for transformation and Gibson assemblies were done in 96-well PCR plates (Eppendorf) using Mastercycler pro S or Mastercycler gradient thermocyclers (Eppendorf).

Heydenreich F.M., Miljuš T., Jaussi R., Benoit R., Milić D, & Veprintsev D.B. (2017). High-throughput mutagenesis using a two-fragment PCR approach. Scientific Reports, 7, 6787.

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Flow Cytometry-based T-cell Receptor Profiling

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T-cell lines were stained with A2-GLC-tetramer-PE for 1h at room temperature. Cells were washed twice, then stained with anti-CD3-PE-Cy7 (#563423), anti-CD8-PerCP-Cy5.5 (#341051), anti-CD14-APC-Cy7 (#560180), anti-CD19-APC-Cy7 (#560177) and LIVE/DEAD Fixable NIR (Molecular Probes, Life Technologies, Eugene, Oregon, USA). Antibodies (their catalog number in brackets) were from BD Biosciences. CD3⁺CD8⁺tetramer⁺dump⁻ single-cells were sorted (FACSAria III, BD) into 96-well PCR plates (Eppendorf, Hamburg, Germany). Analysis of paired CDR3α and CDR3β regions were performed by multiplex-nested reverse transcriptase PCR, then sequencing of TCRα and TCRβ products.^{21 , 22 (link)} Sequences were analyzed according to the IMGT/V-QUEST web-based tool.^{33 (link), 34 (link)}

Nguyen T.H., Bird N.L., Grant E.J., Miles J.J., Thomas P.G., Kotsimbos T.C., Mifsud N.A, & Kedzierska K. (2016). Maintenance of the EBV-specific CD8+ TCRαβ repertoire in immunosuppressed lung transplant recipients. Immunology and Cell Biology, 95(1), 77-86.

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Single B Cell Sorting for Antibody Analysis

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Single human B cells were isolated using a FACSARIA II cell sorter with Diva configuration (BD Biosciences) following tetramer enrichment and cell surface marker staining using human PBMCs as described above. Single B cells from defined subpopulations were sorted according to surface marker expression patterns. Specifically, IB2- or IB3-specific B cells were sorted using side scatter–based doublet discrimination, followed by gating on FVD⁻ CD19⁺ CD20⁺ CD3⁻ CD14⁻ CD16⁻ B cells that bound IB2-PE or IB3-APC tetramers but not PE650 or APC755 tetramers containing isotype control antibodies. We also sorted 576 control FVD⁻ CD19⁺ CD20⁺ CD3⁻ CD14⁻ CD16⁻ CD27⁻ IgM/D⁺ B cells from two individuals that were not stained with an anti-idiotype. Single cells were sorted into individual wells of 96-well PCR plates (Eppendorf) containing 10 µl/well ice-cold lysis buffer containing 0.25 µl (12.5 U) RNaseOUT (Thermo Fisher Scientific), 2.5 µl 5× SuperScript IV First Strand Buffer (Thermo Fisher Scientific), 0.625 µl 0.1 M dithiothreitol (Thermo Fisher Scientific), 0.3125 µl 10% Igepal detergent (Sigma-Aldrich), and 6.625 µl diethyl pyrocarbonate–treated water. Plates were sealed with adhesive PCR plate seals (Thermo Fisher Scientific), centrifuged briefly, and immediately frozen on dry ice before storage at −80°C.

Bancroft T., DeBuysscher B.L., Weidle C., Schwartz A., Wall A., Gray M.D., Feng J., Steach H.R., Fitzpatrick K.S., Gewe M.M., Skog P.D., Doyle-Cooper C., Ota T., Strong R.K., Nemazee D., Pancera M., Stamatatos L., McGuire A.T, & Taylor J.J. (2019). Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes. The Journal of Experimental Medicine, 216(10), 2331-2347.

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Env-Specific B Cell Isolation

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Single human B cells were isolated using a FACSARIA II cell sorter (BD Biosciences) following tetramer enrichment and cell surface marker staining using human PBMCs as described above. Specifically, Env-specific B cells were sorted using SSC-H/SSC-W-based duplet discrimination, followed by gating on CD19⁺ CD20⁺ CD3⁻ CD14⁻ CD16⁻ FVD⁻ B cells that bound HIV-1 Env-PE (or APC) tetramers but not PE650 (or PE594 or APC755) tetramers containing control antigens. Single cells were sorted into individual wells of 96-well PCR plates (Eppendorf) containing 10 μL/well of ice-cold lysis buffer containing 0.25 μL 12.5 U RNase out (Thermo Fisher Scientific), 2.5 μL 5x SuperScript IV First Strand Buffer (Thermo Fisher Scientific), 0.625 μL 0.1M DTT (Thermo Fisher Scientific), 0.3125 μL 10% Igepal detergent (Millipore Sigma), and 6.625 μL DEPC treated water. Plates were sealed with adhesive PCR plate seals (Thermo Fisher Scientific), centrifuged briefly and immediately frozen on dry ice before storage at −80°C.

Steach H.R., DeBuysscher B.L., Schwartz A., Boonyaratanakornkit J., Baker M.L., Tooley M.R., Pease N.A, & Taylor J.J. (2019). Cross-reactivity with self-antigen tunes the functional potential of naïve B cells specific for foreign antigens. Journal of immunology (Baltimore, Md. : 1950), 204(3), 498-509.

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Quantitative Analysis of Chlorinated Tyrosine

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The following materials were purchased from Fisher Scientific (Hanover Park, IL): ammonium bicarbonate, HPLC-grade acetonitrile, HPLC-grade methanol, heat sealing foil, 96-well conical bottom plates, and Optima grade trifluoroacetic acid (TFA). The following materials were purchased from Sigma-Aldrich (St. Louis, MO): Protease Type XIV from Streptomyces griseus (pronase E, lot no. 128K1388, SLBH4482V, SLBD5482V, and SLBP0477V), LCMS grade formic acid, 3-chloro-L-tyrosine (Cl-Tyr), and sodium hypochlorite solution (active chlorine: 4.00–4.99 wt.%). Oasis® HLB 96-well (60 mg, 60 μm) SPE plates were purchased from Waters (Milford, MA). Adhesive PCR foil and 96-well PCR plates were purchased from Eppendorf (Hauppauge, NY). The internal standard ¹³C₆-3-chloro-L-tyrosine (¹³C₆-Cl-Tyr) was purchased from Cambridge Isotopic Laboratories (Andover, MA). Synthetically prepared 3,5-dichloro-L-tyrosine (Cl₂-Tyr) and ¹³C₉,¹⁵N-3,5-dichloro-L-tyrosine (¹³C₉,¹⁵N-Cl₂-Tyr) were purchased from IsoSciences, LLC (King of Prussia, PA). Pooled plasma for preparing quality control (QC) materials was purchased from Tennessee Blood Services (Memphis, TN). All samples used for preparing QC materials were screened by each vendor for hepatitis B, hepatitis C, Treponema pallidum, and HIV in agreement with FDA regulations.

Crow B.S., Quiñones-González J., Pantazides B.G., Perez J.W., Winkeljohn W.R., Garton J.W., Thomas J.D., Blake T.A, & Johnson R.C. (2016). Simultaneous measurement of 3-chlorotyrosine and 3,5-dichlorotyrosine in whole blood, serum, and plasma by isotope dilution HPLC-MS/MS. Journal of analytical toxicology, 40(4), 264-271.

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Single-Cell Analysis of Regulatory T Cells

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CD4+CD25+Foxp3+(RFP+) cells were sorted from a co-culture of WT iTreg and allo-DCs into 96-well PCR plates (Eppendorf) containing 4μl/well of ice-cold 0.5× PBS supplemented with 10 mM DTT, 8U RNAsin (Promega), and 3U RNAse Inhibitor (5Prime). cDNA was synthesized as described [36 (link)], and nested PCR for IL-12Rβ2 and Foxp3 cDNA was performed.
The following primers were used to amplify gene-specific cDNA fragments from the single cells: IL-12Rβ2 outer-down, 5’-CCC TTG GTA TGA CCT TGT TTG TCT GCA AGC-3’; IL-12Rβ2 outer-up, 5’-GAT CCA GGC GAT TGC AAT TCT GAC GAT TGT CA-3’; IL-12Rβ2 inner-down, 5’-AAA AGA AGC CAC CAG TCC CAG TAT G-3’; IL-12Rβ2 inner-up, 5’-CTG ACA GGT CAG ATT GTT TGG TCC A-3’; Foxp3 outer-down, 5’-TTC CAA GGT CGG GAC CTG CGA AGT GGG G-3’; Foxp3 outer-up, 5’-GCA GAA GGT GGT GGG AGG CTG ATC ATG GCT-3’; Foxp3 inner-down, 5’-ACA CCT CTT CTT CCT TGA ACC CCC TG-3’; Foxp3 inner-up, 5’-TCC AGT GGA CGC ACT TGG AGC ACA-3’.
Statistical data were analyzed using Student’s t test.

Sela U., Park C.G., Park A., Olds P., Wang S., Steinman R.M, & Fischetti V.A. (2016). Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses. PLoS ONE, 11(1), e0146412.

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RNA Extraction and qPCR Profiling of Insect Wings

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Between 20 and 30 pupal and newly eclosed (unexpanded) wings were dissected and frozen in Trizol until the RNA extraction process. Total RNA was isolated using Trizol Reagent and cDNA was generated using Bio-Rad’s iScript cDNA synthesis kit.
qPCR analysis was done using iQ™ SYBR Green Supermix by Bio-Rad. The reaction included the mix, cDNA template, and primers at a final concentration of 500mM. The reaction was carried out on the CFX96™ Real-Time PCR Detection system by Bio-Rad. Digital RT-PCR reactions included primers and fluorescent probes specific for each transcript together with Bio-Rad’s ddPCR Supermix for Probes. Droplets were generated using the Droplet Generator (Bio-Rad) with 20ul PCR sample and 70ul Droplet Generator Oil (Bio-Rad). Droplets were transferred to 96 well PCR plates (Eppendorf), heat sealed, and run on an Eppendorf Mastercycler Pro to saturation (45 cycles). Droplets were read on the Droplet Reader (Bio-Rad). Quantasoft software (Bio-rad) assesses the number of positive and negative droplets and applies Poisson statistics to generate an absolute measurement of starting DNA molecules. Rp49 transcript was used as RNA quality control.

Garcia-Hughes G., Link N., Ghosh A.B, & Abrams J.M. (2015). Hid arbitrates collective cell death in the Drosophila wing. Mechanisms of development, 138 Pt 3, 349-355.

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Sorting Antigen-Specific Memory B Cells

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To sort antigen-specific cells, tetramers were generated as described in Franz et al. (52 (link)). A mix of Neutravidin (Neutravidin DyLight650, Thermo Scientific) and biotinylated peptide was incubated in the dark on ice for 30 min. Aggregates were removed by high speed centrifugation. B cells from acute Borreliosis patients were stained for 30 min with a pool of the three peptide tetramers and washed twice with FACS buffer (PBS, 2% FBS). The following antibodies were used to distinguish the different memory B cell subpopulations and to gate out monocytes, T-cells and dead cells: CD14-FITC, CD3-FITC (Immunotools), IgD-BV421, CD27-PECF594 (BD Horizon), CD19-BV605 (BDPharmingen), and live/dead marker. CD20-Biotin (Immunotools) was used as a compensation control for Neutravidin. Single cells were sorted on a FACSAria SORP machine (BD Biosciences) into 96-well PCR plates (Eppendorf) containing 5μl of 0.5% PBS, 10 mM DTT (Invitrogen), and 5U Recombinant RNasin® Ribonuclease Inhibitor (Promega) per well (53 (link)). The plate holder of the sorter was kept at 4°C throughout the sorting procedure. Random, negative and tetramer positive CD19⁺CD27⁺CD14⁻, CD3⁻, Hoechst⁻ B cells were sorted into 96-well plates, which were immediately put on dry ice and stored at −80°C.

Kirpach J., Colone A., Bürckert J.P., Faison W.J., Dubois A.R., Sinner R., Reye A.L, & Muller C.P. (2019). Detection of a Low Level and Heterogeneous B Cell Immune Response in Peripheral Blood of Acute Borreliosis Patients With High Throughput Sequencing. Frontiers in Immunology, 10, 1105.

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Single-cell TCR Sequencing of T Cells

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For human and mouse scTCRseq, T cells were stained as above and sorted at 1 cell/well into 96-well PCR plates (Eppendorf). cDNA was produced using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher) before being subjected to two rounds of semi-nested multiplex PCR^{30 (link)} using PCR master mix (ThermoFisher) and multiplexed human or mouse TCR primer sets as previously described in refs. ⁴³ and ^{30 (link)}. Successfully amplified TCR genes as determined by agarose gel electrophoresis were subjected to Sanger Sequencing using internal TRAC or TRBC primers at Australian Genome Research Facility (AGRF), Melbourne. Sequence data were analysed using the IMGT V-QUEST sequence alignment software^{44 (link)}.

Koay H.F., Gherardin N.A., Xu C., Seneviratna R., Zhao Z., Chen Z., Fairlie D.P., McCluskey J., Pellicci D.G., Uldrich A.P, & Godfrey D.I. (2019). Diverse MR1-restricted T cells in mice and humans. Nature Communications, 10, 2243.

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Synovial Cell Extraction and Characterization

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Synovial tissue was isolated from OA patients who had total knee arthroplasty. The tissue was digested with 1.5mg/mL type 2 Collagenase, 0.7mg/mL hyaluronidase, and 0.3mg/mL DNase I (1–2g tissue/10mL digestion buffer) on a rotating shaker at 270rpm and 37°C for 4 hours. The dispersed cells were either used to seed the wells in 96-well PCR plates (Eppendorf) for single-cell Smart-Seq2 RNA-Seq or to perform flow cytometry analysis. Cells were stained with lineage, c-Kit, FcεRI, CD34, and Integrinβ7 antibodies (BD).

Liu H., Lampe M.A., Iregui M.V, & Cantor H. (1991). Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways.. Proceedings of the National Academy of Sciences of the United States of America, 88(19), 8705-8709.

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