iSAT reactions of 15 μl were set-up as previously described (44 (link)). Briefly, reactions were prepared in polymerase chain reaction tubes with optically clear flat caps and incubated at 37°C in a CFX96 real-time thermal cycler (Bio-Rad). iSAT reactions contained reporter protein plasmids encoding superfolder GFP (sfGFP). Green fluorescence of sfGFP was monitored using the CFX96real-time thermal cycler as (excitation: 450–490 nm, emission: 510–530 nm). Additives were included at the described final concentrations. Specifically, crowding agent (2% PEG-6000) and reducing agent (2 mM DTT) were added to each reaction. iSAT reactions for S150 extracts were optimized for concentrations of magnesium glutamate to maximize reaction productivity and minimize consumption of parts (Supplementary Figure S12 ). sfGFP quantification was performed as previously reported (43 (link)), using measurements of relative fluorescence units (RFU) from CFX96 real-time thermal cycler (BioRad, Hercules, CA, USA) and BioTek Synergy 2 plate reader (Winooski, VT, USA). RFU values were converted to molar concentration using a linear standard curve made in-house by expressing 14C-leucine labeled sfGFP in E. coli PANOx CFPS reactions and relating RFUs to trichloracetic acid precipitable soluble protein yield.
Cfx96 real time thermal cycler
Manufactured by Bio-Rad
Sourced in United States, Australia
The CFX96 real-time thermal cycler is a laboratory instrument designed for conducting real-time PCR (polymerase chain reaction) experiments. It is capable of precisely controlling temperature and monitoring fluorescence signals during the amplification of DNA or RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (18 mentions)
Iscript cdna synthesis kit, by Bio-Rad (11 mentions)
Dnase 1, by Qiagen (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Mirneasy kit, by Qiagen (5 mentions)
Qiazol, by Qiagen (4 mentions)
Ds 11 spectrophotometer, by DeNovix (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Rt2 first strand kit, by Qiagen (3 mentions)
Rneasy plus mini, by Qiagen (3 mentions)
Superscript 3 vilo master mix, by Thermo Fisher Scientific (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Iscript reverse transcription supermix, by Bio-Rad (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Iscript reverse transcriptase, by Bio-Rad (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (3 mentions)
Quickextract solution, by Illumina (3 mentions)
Taqman allelic discrimination assay, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
78 protocols using cfx96 real time thermal cycler
1
In Vitro Synthesis of Fluorescent Protein
2
SARS-CoV-2 Detection by Molecular Assays
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Nasopharingeal swabs were tested by molecular assays to detect the presence of SARS-CoV-2 in the upper respiratory tract of subjects. Two methods were used: the one-step GeneFinder COVID-19 PLUS RealAmp Kit (OSANG Healthcare Co., Ltd., Anyangcheondong-ro, Dongan-gu, Anyang-si, Gyeonggi-do, Korea) and the Allplex 2019-nCoV Assay (Seegene, Seoul, South Korea). The former was used on the ELITe InGenius platform (ELITech, Torino, Italy) and integrated extraction and amplification. The RT-qPCR kit detected in the same PCR reaction the presence of three SARS-CoV-2 targets: the envelope protein (E), the nucleocapsid protein (N) and the RNA-dependent RNA polymerase (RdRp) genes. According to the producer instructions, a gene was considered detected when the cycle threshold (Ct) value resulted ≤43. The latter method detected the same genes and was performed on CFX-96 real-time thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The fluorescence was measured using a Seegene Viewer (Seegene, Seoul, Korea).
3
Evaluating Podocyte Gene Expression
4
SARS-CoV-2 RNA Detection using Allplex 2019-nCoV Assay
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Allplex™ 2019-nCoV Assay (Seegene, Korea), which targets envelope gene of SARS-CoV-2, was used for SARS-CoV-2 RNA detection according to the manufacturer’s instructions. Briefly, 8 μL of extracted RNA was added to 5 μL of 5 × real-time one-step buffer, 5 μL of 2019-nCoV MuDT Oligo Mix (2019-nCoV-MOM), 2 μL of real-time one-step enzyme, and 5 μL of RNase free water. The CFX-96 real-time thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for amplification. The conditions consisted of 1 cycle of 20 min at 50 °C, 1 min at 95 °C and followed by 45 cycles of 15 s at 94 °C and 30 s at 58 °C. The result was analyzed using Seegene Viewer (Seegene, Korea), in which a cycle threshold value (Ct-value) < 40 for all three target genes was defined as a positive result.
5
Supraspinatus Muscle Gene Expression Analysis
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Measurements were performed as previously described.10 (link),12 (link),13 (link) Total RNA was isolated from supraspinatus muscles using a miRNeasy Kit (Qiagen, Valencia, California), reverse transcribed to complementary DNA using the RT2 First Strand Kit (Qiagen) and amplified in a CFX96 Real-Time thermal cycler (Bio-Rad, Hercules, California). Primer sets were purchased from Qiagen. Expression of mRNA transcripts was normalised to the stable housekeeping gene β-actin, using the methods of Schmittgen and Livak.15 (link) Statistical analysis, as described below, was performed on the 2-∆Ct values. A list of measured transcripts is provided in Table I .
6
Quantitative Analysis of Lentiviral Transduction
7
Quantitative Analysis of Lentiviral Transduction
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Total RNA was isolated from Jurkat cells using Trizol reagent followed by purification of RNA with RNeasy Plus Mini (Qiagen) with Turbo DNase (ThermoFisher) in order to limit DNA contamination. First-strand synthesis used Superscript III Vilo Master mix (Invitrogen) with random hexamers. qPCR was performed in 20 μL using SYBR green reagent (Applied Biosystems) with primers designed against gag, gfp, and gapdh for normalization.. Amplification was on a CFX96 Real Time Thermal Cycler (Bio-Rad) using the following program: 95°C for 10 min, then 45 cycles of 95°C for 15 s and 60°C for 60 s. Cells not transduced with Lenti-GFP vector were used as negative control and the housekeeping gene GAPDH was used to normalize expression levels. The primer sequences used were: gag primers (Forward: 5’-GCTGGAAATGTGGAAAGGAA -3’; Reverse: 5’-AGTCTCTTCGCCAAACCTGA -3’), gfp primers (Forward: 5’-GCAGAGGTGAAGTTCGAAGG -3’; Reverse: 5’-CCAATTGGTGTGTTCTGCTG -3’), gapdh primers (Forward: 5’-AGGGCTGCTTTTAACTCTGGT -3’; Reverse: 5’-CCCCACTTGATTTTGGAGGGA -3’).
8
Quantitative Analysis of DAXX Expression
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Total ribonucleic acid (RNA) was extracted from cultured cells using TRIzol reagent (Ambion, Carlsbad, CA, USA) and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (ABI, Foster City, CA, USA). qPCR was carried out with 1 µl cDNA, 10 µl TransStart Top Green qPCR SuperMix (TransGen Co. Ltd, Beijing, China), and primers in 20-µl reaction volume on a CFX96 Real-Time Thermal Cycler (Bio-Rad, Hercules, CA, USA). The following primer pairs were used: 5′-GATACCTTCCCTGACTATGGGG-3′ and 5′-GTAACCTGATGCCCACATCTC-3′ for DAXX; 5′-CCATCGTCCACCGCAAAT-3′ and 5′-GCTGTCACCTTCACCGTTCC-3′ for ACTIN. Relative expression was calculated after normalization to h-actin. Samples were run in triplicate.
9
Quantifying WEEV Envelope Glycoprotein RNA
10
Quantitative SARS-CoV-2 Detection in Cell Cultures
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To evaluate viral replication in cell cultures for the mutational induction assay, the Allplex SARS-CoV-2 Extraction-Free system (Seegene Inc., Seoul, Korea) was used. It consists of a real-time qRT-PCR multiplex assay based on the use of TaqMan probes, which allows simultaneous detection of four target genes, namely E gene, RdRP/S gene and N gene. Sample preparation, reaction setup and analysis were performed accordingly to the manufacturer instructions59 . Briefly, 15 µL of sample were diluted 1:4 in 45 µL of RNase-free water in a 96-well PCR plate and hence 5 µL of the dilution were transferred to another plate with 16 µL of PCR master mix, containing 5 µL of MOM (MuDT Oligo Mixture, with dNTPs, oligos, primers and TaqMan 5’ fluorophore/3’ Black Hole Quencher probes), 5 µL of enzymes, 5 µL of RNase-free water and 1 µL of internal control for every reaction. A positive and a negative control were included. The assay was run on a CFX96 real-time thermal cycler (Bio-Rad, Feldkirchen, Germany). The amplification process includes cDNA denaturation at 95 °C for 10 s, primers annealing at 60 °C for 15 s and elongation at 72 °C for 10 s (44 cycles). Fluorescent signals were acquired after every amplification cycle. Results analysis and targets quantification were performed with 2019-nCoV viewer from Seegene Inc.
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