Genomic DNA was extracted from whole mosquitoes using the DNeasy kit (Qiagen, Hilden, Germany) and used as a template for molecular analyses. The Vgsc genotype at codon 1014 (995 using An. gambiae (s.s.) numbering) (The Anopheles gambiae 1000 Genomes Consortium, 2017 (link)) were determined using a locked nucleic allele (LNA) assay, which detects wild type and kdr mutants serine or phenylalanine (Lynd et al., 2018 (link)). The triple mutation with Cyp6aa1 duplication, Cyp6p4-I236M and ZZB-TE (cytochrome p450-linked ‘Zanzibar-like’ transposable element) was assessed using three independent LNA assays (Njoroge et al., 2021 ). All assays were run on AriaMx Real-Time PCR machine (Agilent, Santa Clara, USA). TaqMan assays were used to genotype Cyp4j5 and Coeae1d (Weetman et al., 2018 (link)). TaqMan assays used a primer/probe mix in addition to 1× sensimix (Bioline) and DNA template (1 μl) in a 10 μl volume reaction with denaturing for 5 min at 95 °C, followed by 40 cycles of denaturing for 15 s at 92 °C and annealing for 1 min at 60 °C. The TaqMan assays were performed on an Agilent MX3005P Real-Time PCR machine.
Mx3005p realtime pcr machine
Manufactured by Agilent Technologies
Sourced in United States
The Mx3005P real-time PCR machine is a laboratory instrument designed for amplifying and detecting specific DNA sequences in real-time. It is capable of performing quantitative polymerase chain reaction (qPCR) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sypro orange, by Thermo Fisher Scientific (2 mentions)
Ariamx real time pcr machine, by Agilent Technologies (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Sypro orange fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Promega (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
6 protocols using mx3005p realtime pcr machine
1
Genetic Markers for Mosquito Resistance
2
Viral RNA Extraction and Influenza A Subtyping
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Viral RNA was extracted from the collected samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All samples were initially tested using standard quantitative reverse transcription polymerase chain reaction (RT-qPCR) specific for the M gene of influenza A viruses [25 (link)]. Positive RNAs were further tested using gene-specific RT-qPCR assays for the hemagglutinin (HA) and neuraminidase (NA) gene segments of the AIV H5N8 subtype [26 (link)]. Viral RNAs were further tested for the presence of infectious bronchitis virus (IBV) [27 (link)] and Newcastle disease virus (NDV) [28 (link)]. All RT-qPCR reactions were performed using Stratagene MX3005P real-time PCR machine (Agilent, Santa Clara, CA, USA). Virus isolation was performed through allantoic fluid inoculation of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs) according to the standard protocols of the OIE diagnostic manual [29 ]. SPF ECEs were obtained from the Nile SPF project (Koom Oshiem, Fayoum, Egypt). The harvested allantoic fluids were tested for virus hemagglutination activity by hemagglutinin assay and verified by using RT-qPCR.
3
Thermal Shift Assay for RIPK2 Inhibitors
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RIPK2 protein at 2 μM concentration was mixed with inhibitor compounds at 10 μM and a 1:1,000 dilution of SyproOrange fluorescent dye (Invitrogen). Fluorescence-based thermal shift assays were performed in an Mx3005p real-time PCR machine (Agilent) as described by Niesen et al. (2007) (link).
4
Thermal Shift Assay for Protein Stability
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Thermal melting experiments were carried out with an Mx3005p realtime PCR machine (Agilent).
Proteins were buffered in 25 mM HEPES (pH 7.5), 500 mM NaCl and were assayed in a 96-wellplate at a final concentration of 2 µM in a 20 µl volume. Inhibitors were added at a final concentration of 10 µM. SYPRO-Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 5000.
Excitation and emission filters were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3°C per minute, and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence was approximated by the equation
where ΔuG is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant, and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein, respectively [58] . The baselines of the denatured and native state were approximated by a linear fit. The observed temperature shifts, ΔTm obs , for each inhibitor were recorded as the difference between the transition midpoints of sample and reference wells containing protein without inhibitor and were determined by nonliner least-squares fit. Measurements were performed in triplicate.
Proteins were buffered in 25 mM HEPES (pH 7.5), 500 mM NaCl and were assayed in a 96-wellplate at a final concentration of 2 µM in a 20 µl volume. Inhibitors were added at a final concentration of 10 µM. SYPRO-Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 5000.
Excitation and emission filters were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3°C per minute, and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence was approximated by the equation
where ΔuG is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant, and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein, respectively [58] . The baselines of the denatured and native state were approximated by a linear fit. The observed temperature shifts, ΔTm obs , for each inhibitor were recorded as the difference between the transition midpoints of sample and reference wells containing protein without inhibitor and were determined by nonliner least-squares fit. Measurements were performed in triplicate.
5
Quantitative RNA Expression Analysis
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Total RNA was extracted from subconfluent cell cultures using an RNAgents kit. The synthesis of cDNA was performed through reverse transcription of 250 ng RNA using 200 IU M-MLVRT, 20 IU RNasin, 0.5 µg oligo dT and 100 µmol/L dNTP in a total volume of 20 µL.
Semi-quantitative RT-PCR was performed with 1.5 IU Taq DNA polymerase in PCR buffer containing 200 µmol/L dNTP, 1.5 mmol/L MgCl 2 and 25 pmol of each primer in a total volume of 25 µL. The PCR primers used and the size of the resulting PCR products are listed in Supplementary Table 1 (see section on supplementary data given at the end of this article). For semi-quantitative real-time RT-PCR, reverse transcription was made with 250 ng of total RNA in 20 µL containing 20 nmol dNTP, 100 ng random primers, 12 IU RNasin and 200 IU M-MLVRT for 1 h 30 min at 37°C. QPCR was performed in duplicate using an Agilent Technologies Mx3005P real-time PCR machine (Agilent Technologies) and the detection was done by the fluorescent dye SYBRGreen with GoTaq qPCR Master Mix (Promega). Expression levels were calculated according to the 2 -ΔCt method normalized to the Actin mRNA expression level.
Semi-quantitative RT-PCR was performed with 1.5 IU Taq DNA polymerase in PCR buffer containing 200 µmol/L dNTP, 1.5 mmol/L MgCl 2 and 25 pmol of each primer in a total volume of 25 µL. The PCR primers used and the size of the resulting PCR products are listed in Supplementary Table 1 (see section on supplementary data given at the end of this article). For semi-quantitative real-time RT-PCR, reverse transcription was made with 250 ng of total RNA in 20 µL containing 20 nmol dNTP, 100 ng random primers, 12 IU RNasin and 200 IU M-MLVRT for 1 h 30 min at 37°C. QPCR was performed in duplicate using an Agilent Technologies Mx3005P real-time PCR machine (Agilent Technologies) and the detection was done by the fluorescent dye SYBRGreen with GoTaq qPCR Master Mix (Promega). Expression levels were calculated according to the 2 -ΔCt method normalized to the Actin mRNA expression level.
6
Thermal Shift Assay for Protein Stability
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Thermal melting experiments were carried out with an Mx3005p realtime PCR machine (Agilent).
Proteins were buffered in 25 mM HEPES (pH 7.5), 500 mM NaCl and were assayed in a 96-wellplate at a final concentration of 2 µM in a 20 µl volume. Inhibitors were added at a final concentration of 10 µM. SYPRO-Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 5000.
Excitation and emission filters were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3°C per minute, and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence was approximated by the equation
where ΔuG is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant, and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein, respectively [58] . The baselines of the denatured and native state were approximated by a linear fit. The observed temperature shifts, ΔTm obs , for each inhibitor were recorded as the difference between the transition midpoints of sample and reference wells containing protein without inhibitor and were determined by nonliner least-squares fit. Measurements were performed in triplicate.
Proteins were buffered in 25 mM HEPES (pH 7.5), 500 mM NaCl and were assayed in a 96-wellplate at a final concentration of 2 µM in a 20 µl volume. Inhibitors were added at a final concentration of 10 µM. SYPRO-Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 5000.
Excitation and emission filters were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3°C per minute, and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence was approximated by the equation
where ΔuG is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant, and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein, respectively [58] . The baselines of the denatured and native state were approximated by a linear fit. The observed temperature shifts, ΔTm obs , for each inhibitor were recorded as the difference between the transition midpoints of sample and reference wells containing protein without inhibitor and were determined by nonliner least-squares fit. Measurements were performed in triplicate.
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