Two pairs of homologous primers (Table 1 ) were designed with DNASTAR 5.0 software in the conserved region of canine (GenBank: AF354265, AF354265, AF354266, AF354267) and mink (GenBank: L07789). All primers used in this study are listed in Table 1 . With the primers, a cDNA fragment was amplified by RT-PCR using the first strand cDNAs as templates. The PCR reaction was performed under the following conditions in a thermal cycle: initial dematuration at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s; annealing at 54°C for 30 s and extension at 72°C for 1 min: and extension at 72°C for 10 min. PCR products were analyzed by electrophoresis in 1% agarose, and purified by Agarose Gel DNA Extraction Kit (Shanghai Sangon Biotech Co. Ltd). The products were cloned by pMD18-T (TaKaRa) and sent to Shanghai Sangon Biotech CO., Ltd. for sequencing.
Agarose gel dna extraction kit
Manufactured by Sangon
Sourced in China
The Agarose gel DNA extraction kit is a laboratory product designed for the extraction and purification of DNA from agarose gel. It provides a method to isolate DNA fragments from agarose gel after electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
One step rt pcr kit, by Takara Bio (2 mentions)
Pmd18 t, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Peasy t1, by Transgene (1 mentions)
Peasy t1 vector, by Transgene (1 mentions)
Peasy t1 cloning vector, by Transgene (1 mentions)
Agarose gel dna extraction kit, by Takara Bio (1 mentions)
7 protocols using agarose gel dna extraction kit
1
Canine and Mink Gene Amplification
2
RT-PCR Detection of Diverse Coronaviruses
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The RNA in the collected feces and drinking-water samples or in the allantoic fluids was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and amplified with the QIAGEN One Step RT-PCR Kit (Qiagen), using a conserved RT-PCR assay designed by ourselves with the primers 5'-GGTTGGGAYTAYCCYAAGTGTGA-3' (upper) and 5'-GAATCIGCCATAWAAACATTRTT-3' (down). The assay amplifies a 545-nucleotide region in the viral 1ab gene, and we have found that this assay can detect some CoVs circulating humans, pigs, chickens, ducks, geese, and pigeons (the data will be published in another paper). The RT-PCR detection was performed in a 25-μL reaction system with incubation at 42°C for 30 min and denaturation at 94°C for 60 s, followed by 30 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 1 min. RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China), and sequenced directly using the ABI 3730xl DNA Analyzer for the following phylogenetic analysis.
3
Genetic Characterization of Avian Coronaviruses
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A conserved RT–PCR assay and DNA sequencing were used to analyze the genetic characteristics of the CoVs circulating in the poultry. In brief, the stored RNAs described above were amplified with the Takara One Step RT–PCR Kit (Takara), using a conserved RT–PCR assay designed in our laboratory, with primers 5′-GGTTGGGATTAYCCWAARTGYGA-3′ (forward) and 5′-YTGTGAACAAAAYTCRTGWGGACC-3′ (reverse). The amplify region is 14,181–14,780 bp in the RDRP gene of the reference sequence (The GenBanK Accession number is NC_001451). The assay amplifies a 600-bp nucleotide region in the viral ORF1ab, and this assay has been shown to detect the main CoVs circulating in animals, including pigs, chickens, ducks, geese, and pigeons. The RT–PCR was performed in a 25 µl reaction system with incubation at 50 °C for 30 min, followed by denaturation at 94 °C for 5 min, and 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s. The PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China), and sequenced directly with the ABI 3730xl DNA Analyzer. The sequences were used in a subsequent phylogenetic analysis.
4
Molecular Cloning of Mink cDNA Fragment
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A pair of homologous primers (Table 1 ) was designed by DNASTAR 5.0 software with respect to the minks (GenBank: L07789.1). With the primers, a cDNA fragment was amplified through RT-PCR using the first strand cDNAs as templates. The PCR reaction was performed under the following conditions in a thermal cycle: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s; annealing at 55°C for 30 s and extension at 72°C for 10 min. Polymerase chain reaction (PCR) products were analyzed by electrophoresis in 1% agarose, and purified by Agarose Gel DNA Extraction Kit (Shanghai Sangon Biotech Co., Ltd.). The products were cloned by Peasy-T1(TransGen) and sent to Shanghai Sangon Biotec Co., Ltd. for sequencing.
5
Cloning and Sequencing of Conserved cDNA Fragment
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A pair of homologous primers (Table 1A ) was designed with DNASTAR 5.0 software in the conserved region of Gallus (EU477527.1) and Taeniopygia guttata (XM_002189232.1). All primers used in this study are listed in Table 1 . With the primers, a cDNA fragment was amplified by RT-PCR using the first strand cDNAs as templates. The PCR was performed at 94°C for 5 minutes, followed by 30 cycles at 94°C for 45 seconds, 58°C for 40 seconds and 72°C for 1 minute, with a final extension at 72°C for 10 minutes, and then stopped at 4°C. Polymerase chain reaction products were detected by electrophoresing 5 µl aliquots through 1% agarose in 1 × TAE and purified by Agarose Gel DNA Extraction Kit (Shanghai Sangon Biotech Co., Ltd.). The products were cloned by into pEASY-T1 vector (TransGen) and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Two pairs of specific primers (Table 1 : 3'RACE 1 and 2) were designed according to the obtained sequences. In the 3'RACE and the second pair for the second round PCR products were analysed by electrophoresis in 1% agarose then purified and cloned into pEASY-T1 vector, detected and sequenced as mentioned above.
6
Characterization of Duck Coronavirus Genome
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We isolated DdCoV/DK/Guangdong/27/2014 which is abbreviated as DdCoV/GD/2014 from ducks in 2014. The whole genome of DdCoV/GD/2014 was amplified through RT-PCR with a series of primers (see S1 Table ). The primers were designed according to the conserved regions in the genomes of the CoVs in genus Gammacoronavirus and the regions having been sequenced through NGS [12 (link)]. The RT-PCR reactions were performed in a 50-μl reaction system with incubation at 42°C for 30 min and denaturation at 94°C for 60 s, followed by 30 cycles at 94°C for 30 s, 50–55°C (largely depending on the Tm values of the primers) for 30 s, and 72°C for 1–4 min (depending on the length of the amplicons). RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China). The amplicons were purified using an agarose gel DNA extraction kit (Takara, Dalian, China) and ligated into the pEASY-T1 cloning vector (TransGen, Beijing, China). Positive clones were sequenced by the ABI 3730xl DNA Analyzer using the pair of M13 primers from both senses. Sequences were assembled and edited manually to generate the whole genome sequence which was further compared to those of IBVs. The DdCoV genome sequence was annotated manually.
7
Conserved RT-PCR and Sequencing for Avian CoV Surveillance
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A conserved RT-PCR and sequencing was used to analysis the genetic characteristics of CoVs circulating in poultry.
In brief, the RNA stored above were amplified with the Takara One Step RT-PCR Kit (Takara), using a conserved RT-PCR assay designed by our lab with the primers 5'-GGTTGGGATTAYCCWAARTGYGA-3' (upper) and 5'-YTGTGAACAAAAYTCRTGWGGACC-3'(down). The assay amplifies a 550 bp-nucleotide region in the viral 1ab gene, and this assay has been proved to detect main CoV circulating in animals, including pigs, chickens, ducks, geese, pigeons, etc. The RT-PCR detection was performed in a 25-μl reaction system with incubation at 50 for 30 min and denaturation at 94 for 5 min, followed by 30 cycles at 94 for 30 s, 50 for 30 s and 72 for 45 s. RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China), and sequenced directly using the ABI 3730xl DNA Analyzer for the following phylogenetic analysis.
In brief, the RNA stored above were amplified with the Takara One Step RT-PCR Kit (Takara), using a conserved RT-PCR assay designed by our lab with the primers 5'-GGTTGGGATTAYCCWAARTGYGA-3' (upper) and 5'-YTGTGAACAAAAYTCRTGWGGACC-3'(down). The assay amplifies a 550 bp-nucleotide region in the viral 1ab gene, and this assay has been proved to detect main CoV circulating in animals, including pigs, chickens, ducks, geese, pigeons, etc. The RT-PCR detection was performed in a 25-μl reaction system with incubation at 50 for 30 min and denaturation at 94 for 5 min, followed by 30 cycles at 94 for 30 s, 50 for 30 s and 72 for 45 s. RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China), and sequenced directly using the ABI 3730xl DNA Analyzer for the following phylogenetic analysis.
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