For routine testing, viral RNA was extracted using the High Pure Viral Nucleic Acid kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Two RT–PCR primer sets were used, a universal IBV RT–PCR generating a fragment of approximately 350 base pairs of the S1 gene with primers XCE1 + and XCE3- (Cavanagh et al., 1999 (link)) and a genotype-specific RT–PCR for D1466-like strains using forward primer 5′-TACRggMAATTTTACTgATgg-3′ and reverse primer 5′-CTgACTgCTTACAAgAACC-3′. Both RT-PCRs were performed using a VeritiTM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) in combination with the Qiagen one-step RT–PCR kit (Qiagen, Hilden, Germany) under the following conditions: reverse transcription reaction for 30 min at 50°C; denaturation 15 min at 95°C, followed by 45 cycles with 30 s at 95°C, 30 s at 50°C and 60 s at 72°C. The S1 amplicons were separated on a 1% agarose gel, and visualized with ethidium bromide staining and an ultraviolet light transilluminator. The purified amplicons were sequenced bidirectionally using Sanger sequencing with both the XCE1+ and XCE3-primers (BaseClear, Leiden, the Netherlands). Consensus sequences were constructed using MEGA7.0 (Kumar et al., 2016 (link)).
Verititm thermal cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Veriti Thermal Cycler is a laboratory instrument used for DNA amplification through the polymerase chain reaction (PCR) process. It precisely controls the temperature and duration of the various steps involved in the PCR procedure to facilitate the replication of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Epitect pcr control dna set, by Qiagen (2 mentions)
Streptavidin coated sepharose beads, by GE Healthcare (2 mentions)
Pyromark gold q96 reagent, by Qiagen (2 mentions)
Pyromark assay design version 2, by Qiagen (2 mentions)
Pyromark q96 id system, by Qiagen (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
Gelred, by Biotium (1 mentions)
Bigdyetm terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Sequencing analysis software v5, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Agencount ampure xp beads, by Beckman Coulter (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Multiplex pcr master mix, by Qiagen (1 mentions)
Abi prism 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Milli q, by Merck Group (1 mentions)
T100tm thermal cycler, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
11 protocols using verititm thermal cycler
1
Detecting Avian Coronavirus Strains
2
Murine PC/5TGM1 Gene Expression Analysis
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Total RNA was extracted from isolated murine PCs/5TGM1 cells and reverse transcribed as described above. Separate PCRs for Glipr1 and Actb (see primer sequences above) was performed using AmpliTaq GoldTM DNA Polymerase (Thermo Fisher Scientific) on a VeritiTM Thermal Cycler (Thermo Fisher Scientific). The PCR products from the cDNA were then visualised by agarose gel electrophoresis using a 2% (w/v) agarose gel containing 1:10,000 GelRed® (Biotium).
3
Genotyping of Vitamin D Receptor Polymorphisms
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The DNA was amplified by polymerase chain reaction (PCR) using specific primers for rs7975232, rs1544410, rs731236, and rs2228570. Primer sequence descriptions of the four single nucleotide polymorphisms (SNPs) are shown in Table S1 . The PCR reaction system included 0.5 µL primer, 2.5 µL PCR buffer, 0.75 µL MgCl2, 2.0 µL dNTP, 0.2 µL Taq polymerase, 1.0 µL DNA, and 17.55 µL water to a final reaction volume of 25 µL. PCR was carried out at Thermo Fisher Scientific VeritiTM Thermal Cycler(Thermo Fisher Scientific Inc., Wilmington, DE, USA) using the following procedures: 1 cycle of 95 °C denaturation for 10 min, 35 cycles of 95 °C denaturation for 15 s, annealing temperature adjusted by primer for 30 s, and 72 °C extension for 1 min 30 s. Thereafter, this PCR product went through sequencing reaction using BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Wilmington, DE, USA) in ABI PRISM 3130 Genetic Analyzer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The produced nucleotide sequences were analyzed with the Sequencing Analysis Software v5.2 (Thermo Fisher Scientific Inc., Wilmington, DE, USA).
4
DNA Barcoding: ITS2 Amplification
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PCR amplification was performed according to the DNA barcoding protocol in the Chinese Pharmacopoeia on an Applied Biosystems VeritiTM Thermal Cycler (Thermo Fisher Scientific). Individual amplifications of ITS2 were carried out in 25 uL total volumes, including 10 ng template DNA and 2× Master Mix (AmpliTaq gold fast PCR MM, Thermo Fisher Scientific). The PCR annealing temperature was increased to 58 °C, and 25 cycles were run.
The PCR products were purified with Agencount Ampure XP beads (Beckman) and the PCR size and concentrations were measured on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Folsom, CA, USA) with the Qubit platform.
The PCR products were purified with Agencount Ampure XP beads (Beckman) and the PCR size and concentrations were measured on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Folsom, CA, USA) with the Qubit platform.
5
Optimized SSR Analysis Protocol
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To perform SSR analysis, 39 microsatellite markers were used under optimized conditions (Table 4 ). Molecular markers were amplified using multiplex (Table 5 ) PCR in an automated thermocycler VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The 10 μL reaction mixture contained 2× QIAGEN Multiplex PCR Master Mix (QIAGEN, Hilden, Germany), 5 μM of each primer, and 100 ng of DNA. The amplification profile consisted of the initial denaturation of the template DNA and HotStarTaq DNA polymerase activation at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 55–60 °C (depending on the primer) for 90 s, and product extension at 72 °C for 60 s. The final extension of the amplicons was performed at 60 °C for 30 min. The PCR products were checked by electrophoresis in an agarose gel (2%), and then separated using capillary electrophoresis in an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The SSR data were processed using GeneMapper software v6 (Applied Biosystems, Foster City, CA, USA).
6
Comprehensive Plant DNA Barcoding Protocol
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A PCR master mix reaction comprised 3 μl of 10 × PCR buffer, 2.4 μl of 2.5 mM dNTPs, 3 μl of 2.5 mM MgCl2, 1.5 μl of 10 μM of each primer, 3 μL of DNA template, 0.2 μl of 5 U/μL Taq DNA polymerase, and double distilled Milli-Q (Merck Millipore) Ultrapure Water filling up to 30 μl. The PCR amplification was performed using VeritiTM Thermal Cycler (Applied Biosystems, USA) or T100TM Thermal Cycler (Bio-Rad, USA). PCR amplification protocols at the psbA-trnH, rpoC1, rps14, rbcL and ITS2 regions are presented in Appendix B . Positive and negative controls were applied for each PCR amplification. Positive control was the DNA of Datura stramonium L. extracted by the first author.
Gel electrophoresis was conducted for visualizing amplicons with 1.5% 1x TAE gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific) under blue light. Gel purification was performed using a DNA gel purification kit (Biomed, China). Sanger sequencing was performed by BGI Hong Kong. Subsequently, Consensus sequences between the forward and reverse sequences were generated and identities elucidated by blast search on GenBank. ClustalW multiple alignment with the combined DNA sequences was conducted using BioEdit version 7.0.5.3 (10/28/05) to assess the nucleotide differences between species.
Gel electrophoresis was conducted for visualizing amplicons with 1.5% 1x TAE gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific) under blue light. Gel purification was performed using a DNA gel purification kit (Biomed, China). Sanger sequencing was performed by BGI Hong Kong. Subsequently, Consensus sequences between the forward and reverse sequences were generated and identities elucidated by blast search on GenBank. ClustalW multiple alignment with the combined DNA sequences was conducted using BioEdit version 7.0.5.3 (10/28/05) to assess the nucleotide differences between species.
7
Quantitative PCR Analysis of Gene Expression
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RNA was extracted from the samples using Qiagen RNeasy Plus Mini Kit (74134, Qiagen, Hilden, Germany) and genomic DNA was removed using RNase-Free DNase Set (79254, Qiagen). RNA purity and concentration were quantified using Epoch Take3 microvolume plate (Biotek) and cDNA was synthesized using TranscriptME RNA kit (RT31, Blirt, Gdansk, Poland). PCR was carried out with 25 ng of cDNA templates using 2X PCR TaqNova-RED (RP85T, Blirt), which included all the components needed to perform cDNA amplification. The primers used are listed in Table 1 . The PCR (VeritiTM Thermal Cycler, Applied Biosystems, Foster City, CA, USA) was carried out under the following conditions: 5 min at 94 °C followed by 30 cycles of 15 s at 94 °C, 30s at 55 °C, and 15 s at 72 °C, and a final extension step of 5 min at 72 °C. Finally, DNA electrophoresis in 2% agarose gels was performed with PCR products to determine the relative expression of the selected genes.
8
Quantifying Gene Expression via qRT-PCR
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For cDNA syntesis, 1 μg of total RNA were reverse transcribed using high capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s protocol on the VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, United States). Target gene expression was determined via quantitative Real-Time PCR analysis using Taqman Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, United States) on 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, United States). The expression values of the genes of interest (APC; Hs01568269) in each of the transfected cells of constructs plasmid were normalized to the expression value of the endogenous control, GAPDH (catalog no. 1391084). Relative fold change of the targeted gene levels between the transfection groups were determined by the 2-ΔΔCT. All experiment reactions were performed in triplicates.
9
Quantifying DNA Methylation Levels in PLD3
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Genomic DNA was isolated from frozen hippocampal tissue by phenol-chloroform method [22 (link)]. Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen, Redwood City, CA, USA) according to the manufacturer’s protocol. Primers to amplify and sequence two promoter regions of PLD3 were designed with PyroMark Assay Design version 2.0.1.15 (Qiagen) (Additional file 1 : Table S2), and PCR reactions were carried out on a VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Next, 20 μl of biotinylated PCR product was immobilized using streptavidin-coated sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, USA) and 0.3 μM sequencing primer was annealed to purified DNA strands. Pyrosequencing was performed using the PyroMark Gold Q96 reagents (Qiagen) on a PyroMark™ Q96 ID System (Qiagen). For each particular cytosine-phosphate-guanine dinucleotide (CpG), methylation levels were expressed as percentage of methylated cytosines over the sum of total cytosines. Unmethylated and methylated DNA samples (EpiTect PCR Control DNA Set, Qiagen) were used as controls for the pyrosequencing reaction.
10
DNA Methylation Analysis of Neural Progenitor Cells
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Genomic DNA was isolated from frozen cell pellets of basal NPCs and control or Aβ peptide treated NPCs incubated in differentiation media for 9, 19, or 29 days by using the FlexiGene DNA Kit (Qiagen, Redwood City, CA, USA). Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Primer pairs to amplify and sequence the chosen CpG genomic positions were designed with PyroMark Assay Design version 2.0.1.15 (Qiagen) (Supplementary Table S1 ) and bisulfite PCR reactions were carried out on a VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Next, 20 μL of the biotinylated PCR product was immobilized using streptavidin-coated Sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, USA) and 0.4 μM of sequencing primer annealed to purified DNA strands. Pyrosequencing was performed using PyroMark Gold Q96 reagents (Qiagen) on a PyroMark™ Q96 ID System (Qiagen). For each particular CpG, DNA methylation levels were expressed as the percentage of methylated cytosines over the sum of total cytosines. Unmethylated and methylated DNA samples (EpiTect PCR Control DNA Set, Qiagen) were used as controls for the pyrosequencing reaction.
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