The genotypes of parental Il6rafl/fl and Pdgfra-Cre mice, as well as their offspring, were determined using PCR to evaluate the zygosity of floxed Il6ra alleles and the expression of Cre in genomic DNA isolated from ear punch tissue as previously described38 (link). Briefly, the tissue was lysed in 0.1 M NaOH with 0.2 mM EDTA for 1 h at 95 °C. The solution was neutralized with 40 mM Tris (pH 5.0), and residual tissue was pelleted by centrifugation. PCR was performed using SsoAdvanced Universal SYBR Green Supermix and Bio-Rad CFX Connect Thermocycler (Bio-Rad Laboratories, Hercules, CA), and allele-specific primers were purchased from Integrated DNA Technology (San Jose, CA, USA). PCR product length for each reaction was verified by 1.5% agarose gel electrophoresis using a GeneRuler 1 kb Plus DNA Ladder (SM1332, Thermo Scientific, Waltham, MA, USA), with bands visualized using a ChemiDoc Imaging System (Bio-Rad). The wildtype and floxed Il6ra alleles were detected by PCR amplification of 109 bp and 123 bp fragments, respectively, while Cre expression was determined by PCR amplification of a 635 bp fragment. Floxed mice (Il6rafl/fl) were distinguished from heterozygous floxed (Il6rawt/fl) littermates by Il6rafl/fl lacking a WT band on the gel. Genotyping primer sequences for Il6ra were provided by The Jackson Laboratory as summarized in Supplemental Table 1 39 .
Cfx connect thermocycler
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The CFX Connect thermocycler is a reliable and efficient instrument designed for DNA amplification and analysis. It features a compact design, intuitive software, and precise temperature control to ensure consistent and accurate results. The core function of the CFX Connect is to perform the thermal cycling required for various molecular biology techniques, such as polymerase chain reaction (PCR).
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Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (15 mentions)
Rneasy mini kit, by Qiagen (11 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Iq sybr green supermix, by Bio-Rad (6 mentions)
Rneasy micro kit, by Qiagen (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Ssoadvanced universal sybr green qpcr supermix, by Bio-Rad (4 mentions)
Ssoadvanced sybr green master mix, by Bio-Rad (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Itaq universal sybr green supermix, by Bio-Rad (4 mentions)
Random primer, by Promega (4 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (3 mentions)
Nanodrop 2000 spectrometer, by Thermo Fisher Scientific (3 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (3 mentions)
Primepcr technology, by Bio-Rad (3 mentions)
Sybr green supermix, by Bio-Rad (3 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (2 mentions)
64 protocols using cfx connect thermocycler
1
Genotyping Il6ra Floxed and Pdgfra-Cre Mice
2
Genotyping of Mouse Retinal Degeneration Genes
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The presence of wildtype alleles of Pde6b, Crb1, and Gpr179 genes or their rd1, rd8, and nob5 mutant alleles, respectively, was evaluated in wildtype (C57BL/6J), B6;SJL-Il6ratm1.1Drew/J, C57BL/6-Tg(Pdgfra-cre)1Clc/J, and MGC-specific Il6ra−/− mice using allele-specific PCR of genomic DNA as previously described45 (link),50 (link)–54 . PCR reactions were performed with SsoAdvanced Universal SYBR Green Supermix and Bio-Rad CFX Connect Thermocycler (Bio-Rad Laboratories), and PCR product length was determined via 1.5% agarose gel electrophoresis using a GeneRuler 1 kb Plus DNA Ladder (SM1332, Thermo Scientific, Waltham, MA) and ChemiDoc Imaging System for gel visualization (Bio-Rad). Primer sequences used for PCR amplification are summarized in Supplemental Table 1 .
3
Validating Gene Expression by qRT-PCR
4
RNA Extraction and qPCR Analysis
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Adult leaf or whole-seedling tissue was ground to a powder in liquid nitrogen and 0.1 g was mixed with 1 ml of TRIzol reagent (Invitrogen). Total RNA was extracted by chloroform, then isopropanol at room temperature before overnight precipitation in 100% EtOH at _ 20°C. The RNA pellet was resuspended in diethyl pyrocarbonate H 2 O and a total of 10 µg was treated with TURBO DNase (Invitrogen). For reverse transcription PCR experiments, cDNA was synthesized from 2 µg of total purified RNA using an oligo(dT)15 primer and Moloney murine leukemia virus reverse transcription (Promega), following the manufacturer's protocol. qPCR was performed with 5 µl of 20× diluted cDNA and a primer concentration of 1 µM in a 20-µl reaction with Brilliant III Ultra-Fast SYBR GREEN master mix (Agilent) with a BioRad CFX Connect thermocycler (Bio-Rad). Unless otherwise stated, transcript levels of target were normalized to the housekeeping gene SAND.
5
Quantitative Real-Time PCR for Gene Expression Analysis
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Total RNA was extracted using the RNeasy Mini Kit (Qiagen) due to the manufacturer's instructions. The RNA concentration was photometrically assessed using the Nanodrop2000 spectrometer (Thermo Scientific). Two micrograms of RNA were utilized to synthesize complementary cDNA single strands using M-MLV reverse transcriptase (Promega) and random hexameric primers. Quantitative real time PCR was carried out using the Sso Advanced SYBR Green Supermix (BioRad) in a CFX Connect Thermocycler (BioRad). A total of 10 ng cDNA and 10 pmol per primer were used in each qPCR reaction. The relative quantifications were normalized to the endogenous housekeeping genes β-actin and β-2-microglobulin. Calculation of normalized relative gene expression was performed by supplied software of the CFX Connect Real-Time PCR Detection System (Bio-Rad). The figures show data from three independent experiments represented as mean ± SD. An unpaired student t test was performed to calculate statistical significance. The Primer sequences can be found in Supplementary Table S1 .
6
Quantifying CYP1A1 Induction Dynamics
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Dose–response curves for the induction of CYP1A1 mRNA expression by TCDD exposure were determined after 24 h of exposure using quantitative reverse transcription PCR (qRT-PCR). AHR mRNA to assess the level of overexpression, zeocin resistance gene (ZEO) expression to quantify transfection efficiency, as well as β-actin (ACTB) mRNA expression levels were determined in sample aliquots measured in parallel. All qRT-PCR data, including background AHR and ARNT mRNA levels measured in HeLa and HepG2 cells, were normalized for differences in cDNA input based on ACTB mRNA expression. Harvesting of cells from 96-well cell culture plates and qRT-PCR was carried out using the Ambion Power SYBR Green Cells-to-CT Kit (Thermofisher Scientific 4402954). Alternatively, for small-scale experiments and nontransfected HeLa and HepG2 cells, total RNA was isolated using TRIzol reagent (Invitrogen, Fisher Scientific), reverse transcribed into cDNA using the iScript cDNA Synthesis Kit, and quantitative PCR was carried out using IQ SYBR Green Supermix (both from Bio-Rad Laboratories B.V., Veenendaal, the Netherlands). All primers and annealing temperatures used are listed in supplementary material S8 , Supplementary Material online. Melt-curve analysis was performed to assure a single PCR product of the expected melting temperature. A Bio-Rad CFX Connect thermocycler was used throughout.
7
Quantitative Real-Time PCR Analysis
8
Quantifying Gene Expression in Mice
9
RNA Isolation and qRT-PCR Analysis
10
Quantifying Gene Expression Changes
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Cells were plated at a density of 187 000 cells in 60 mm tissue culture plates and treated with 1 μg/ml of doxycycline for 72 h. Cells were harvested with Trizol® (Invitrogen) and RNA was isolated according to manufacturer's instructions. Isolated RNA was further purified using the RNeasy® kit (Qiagen). Two micrograms (2 μg) of isolated RNA was converted to cDNA using Verso™ cDNA synthesis kit (ThermoFisher). cDNA was diluted ∼4-fold with water and qRT-PCR was performed with maxima SYBR green master mix (Thermo) and run on the BioRad CFX Connect™ thermo cycler. Six biological replicates were performed in technical triplicate. Data was normalized to the primer efficiency and B2M expression (39 (link)).
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