Two pairs of specific primers (Table 2 ) designed based on the complete genomic sequence of PEDV strain CV777 (AF353511) were used to amplify the whole ORF3 and S1 genes of the PEDV strains identified in this study. The PCR reactions were performed in a 50-μl mixture consisting of 25 μl of 2 × Taq PCR Master Mix (TsingKe Co, Ltd., People’s Republic of China), 2 μl of the cDNA sample, 2 μl of each primer (10 pmol), and sterilized water in a thermal cycler (Biometra) with the following program: 94 °C for 5 min (first denaturation), 35 cycles of 94 °C for 30 s (denaturation); 56 °C for 30 s (annealing), and 72 °C for 30 s or 2 min (for the complete ORF3 or S1 gene, respectively) (extension); followed by 7 min at 72 °C (final extension). The purified PCR products were cloned into the pUCm-T vector and then sequenced. The sequences of these novel PEDV strains were submitted to the NCBI database (GenBank accession numbers MK135440-MK135453 and MK135454-MK135467 for the S1 and ORF3 genes, respectively).
Taq pcr master mix
Manufactured by Tsingke
Sourced in China
The 2 × Taq PCR Master Mix is a pre-mixed solution that contains all the necessary components for performing Polymerase Chain Reaction (PCR) amplification, including Taq DNA polymerase, dNTPs, and reaction buffers. This master mix is designed to simplify the PCR setup process and improve reproducibility.
Automatically generated - may contain errors
4 protocols using taq pcr master mix
1
PEDV Strain Identification and Sequencing
2
Antibiotic Resistance Gene Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total genomic DNA was extracted using a kit (Tiangen Biotech, China). All genomic DNA solutions were stored at -20℃. According to previous studies, 6 antibiotic resistance genes (ARGs), 13 mobile genetic elements (MGEs) and gene cassettes were selected and detected by polymerase chain reaction (PCR)19 (link)–22 (link). PCR assays were carried out in 25 μL volumes containing 2 μL template DNA, 12.5 μL 2 × Taq PCR Master Mix (Tsingke, China), 8.5 μL ddH2O (Solarbio, China) and 1 μL each primer. The PCR products were separated by gel electrophoresis in a 1.5% agarose gel stained with GoldView™ (Sangon Biotech, China), visualized under ultraviolet light and photographed using a gel documentation system (BioRad, USA). The primers and amplification conditions used have been previously described in Table S1 23 (link),24 (link).
3
DCTN1 Gene Exon Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from whole blood using standard protocols (QIAGEN, Valencia, California, USA). A total of 24 pairs of primers were designed for PCR amplification of the 32 protein-coding exons, including intron-exon boundaries, of DCTN1 (GenBank NM_004082.4). PCR was performed in a total volume of 25 μl, which consisted of 20 ng of genomic DNA, 10 pmol of each primer, 12.5μl of 2×Taq PCR Master Mix (Tsingke Biotechnology Co., Ltd., Beijing, China) and 9.5μl of deionized water. The thermocycling conditions were as follows: initial denaturation at 98°C for 2 min, 30 cycles at 98°C for 10 s, 57–65°C for 15 s (the annealing temperature ranged from 57°C to 65°C depending on the primer pair, see supplementary material), and extension at 72°C for 15 s, and a final extension at 72°C for 5 min. We used agarose gel electrophoresis and magnetic beads cleanup method for isolating and purifying PCR products. BigDye Terminator v3.1 Cycle Sequencing Kit and 3730XL DNA Analyzer was used for the amplification of PCR products and Sanger sequencing. All products were sequenced at Tsingke Biotechnology Co., Ltd. Each identified mutation was confirmed by both forward and reverse sequencing, and all mutated sequences were re-amplified and re-sequenced.
4
Isolation of Flavonoid Biosynthesis Genes in Fig
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR primers for isolation of CHS, CHI, F3H, F3’H, DFR and UFGT were designed based on the six complete fig structural gene sequences predicted by our RNA-Seq database (Additional file 1 : Table S1). The full-length gene sequences were cloned from the fig ‘Zibao’ T4 peel cDNA library. PCR was performed in a 20-μL reaction system containing 1 μL first-strand cDNA, 1 μL each of 10 μM forward and reverse primers, 7 μL DEPC-treated water, and 10 μL of 2× Taq PCR MasterMix (Tsingke, Beijing, China). The PCR conditions were as follows: initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis on 1.0% agarose gels, and purified using an agarose gel purification extraction kit (Axygen, Corning, NY, USA), ligated into the pMD19-T vector (TaKaRa, Dalian, China), transformed into E. coli DH5α cells, and positive clones were selected for sequencing (Tsingke Biological Technology Co. Ltd., Beijing, China). The structural gene sequences were analyzed by protein family searches using BLAST in NCBI (http://www.ncbi.nlm.nih.gov/ ). Sequence alignment was performed using ClustalX software version 1.8331 (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/#G ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!