The RNA of PRRSV JXA1 was reverse transcribed to cDNA using a PrimeScript RT Master Mix (Takara Biotech Co., Ltd., Beijing, China). The cDNA was used as the template for PCR amplification of the PRRSV ORF6 gene with the primer pair ORF6 F1/ORF6 R1 (Table 1 ). PCR was conducted using a 2 × Rapid Taq Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) with the following reaction system: 2 × Rapid Taq Master Mix 25 μL, cDNA template 2 μL, forward primer (10 mM) 2 μL, reverse primer (10 mM) 2 μL, ddH2O 19 μL. PCR program was as follows: 95 °C for 3 min (predenaturation); 30 cycles of 95 °C for 15 s (denaturation), 58 °C for 15 s (annealing), and 72 °C for 15 s (extension); 72 °C for 5 min (final extension). The amplification product was purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and ligated to pUCm-T Vector (Beyotime Biotech Co., Ltd., Shanghai, China). The concentration of the resulting plasmid pUCm-ORF6 was measured, then the pUCm-ORF6 copy number was calculated as in a previous study [16 (link)].
Rapid taq master mix
Manufactured by Vazyme
Sourced in China
2× Rapid Taq Master Mix is a ready-to-use solution containing Taq DNA Polymerase, dNTPs, buffer, and other necessary components for PCR amplification. It is designed to provide a fast, efficient, and reliable PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Aceq qpcr sybr green master mix, by Vazyme (4 mentions)
Phanta max master mix, by Vazyme (4 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (3 mentions)
Minibest viral rna dna extraction kit, by Takara Bio (3 mentions)
Novaseq 6000 platform, by Illumina (3 mentions)
Pucm t vector, by Beyotime (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (2 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (2 mentions)
Hiscript 3 1st strand cdna synthesis kit, by Vazyme (2 mentions)
Clonexpress multis one step cloning kit, by Vazyme (2 mentions)
Fastpure plasmid mini kit, by Vazyme (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (2 mentions)
Pmd18 t vector, by Takara Bio (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Tianamp stool dna kit, by Tiangen Biotech (1 mentions)
95 protocols using rapid taq master mix
1
Cloning and quantification of PRRSV ORF6
2
Molecular Marker Development for Wheat Gene
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After analyzing the TaBADH-A1 gene sequence polymorphism in Taishan 1, Bima 4, Zhongmai 9, Lantian 23, and 15 wheat cultivars based on sequences from the database, the molecular marker 6 AM was developed based on the insertion/deletion (indel) of 254 bp in the third intron region. Primers for the molecular marker 6 AM were designed as 5′-CGCAAGTCAATGGGCTGTGTAT-3′ and 5′-CAAGACAGGAGGGGAATGGGAAAT-3′. The PCR system components were as follows: 10 μl of 2× Rapid Taq Master Mix, 7 μl ddH2O, 1 μl 6 AM-F (10 μM), 1 μl 6 AM-R (10 μM), and1 μL DNA (300 ng·μL−1; Rapid Taq Master Mix, Vazyme). The PCR program for 6 AM comprised a cycle at 95°C for 3 min, 95°C for 15 s, 62°C for 15 s, and 72°C for 15 s, followed by 34 additional cycles of 95°C for 15 s, 62°C for 15 s, and 72°C for 15 s, and finally 72°C for 5 min. The PCR products were separated on 2% agarose gel. The primer pairs were located by PCR amplification using the nulli-tetrasomic lines of Chinese Spring as a template. The validity of the 6 AM primers was verified using PCR-based amplified fragment length polymorphism. Genotyping was performed in population 1, 2, 3, and 4 using the 6 AM molecular marker to characterize the function of alleles.
3
Quantifying Genomic Edits via Sanger Sequencing
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Target genomic sites were PCR amplified using the lysate of HEK293T cells and porcine embryos by the Rapid Taq Master Mix (Vazyme), with primers flanking each examined target site. Sanger sequencing was then performed by IGE Biotechnology (Guangzhou, China), and the ratios of C-to-T and A-to-G conversions were calculated by the EditR software48 (link) (https://moriaritylab.shinyapps.io/editr_v10/ ). The primers used to amplify the target genomic sequences are listed in Supplementary Data 2 .
4
Genomic DNA Extraction and PCR Amplification Protocol
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Genomic DNA was extracted using TIANamp Stool DNA Kit and TIANamp Genomic DNA Kit (TIANGEN BIOTECH, Beijing) for fecal samples and blood/tissue samples, respectively. The 20 μl PCR reaction system included 10 μl Mix (2 × Rapid Taq Master Mix, P222-AA, VAZYME BIOTECH, Najing), 8 μl dd H2O, 0.5 μl each primer (25uM solution) and 0.5 ~ 1 μl template DNA (about 2 U). The amplification protocols were carried out as follows: initial denaturation 3 min at 95 ℃, 40 cycles (15 s at 95 ℃, 15 s at 51 ℃, 15 s at 72 ℃), final elongation of 3 min at 72℃. PCR products were visualized on a 3% agarose gel and further capillary electrophoresis on ABI 3,100 genetic analyzer. Genotyping data were obtained by GeneMapper 4.0 (Sangon Biotech, Shanghai; and Tsingke Biological Technology, Beijing).
5
Molecular Detection and Characterization of 'Candidatus Liberibacter asiaticus' in Citrus Leaves
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qPCR was used to detect the CLas bacterium in all leaf samples. Real-time PCR was used to determine the cycle threshold (CT) value by employing previously described primer sets and probes (42 (link)). Following the CLas-infected citrus DNA samples testing, the CLas-positive samples were used to amplify seven hypervariable genomic regions using the primer sets (PS) (Table 3 ). The qPCR procedure was performed in 25-μL mixtures containing 10 μL of 2× Rapid Taq Master Mix (Vazyme, China), 1 μL forward and reverse primers, 2 μL of template DNA, and 11 μL of H2O using a LightCycler 96 real-time PCR system (Roche, USA). The PCR cycles were programmed with an initial denaturation step of 94°C for 5 min, followed by 40 cycles of 95°C for 30 s, 56 to 60°C for 30 s, and 72°C for 30 s, based on the primer sets used. After the last cycle, a final extension of 72°C was performed for 10 min. Following confirmation, amplification products were stained with ethidium bromide under a UV illuminator on 2% agarose, purified, and sequenced using the amplification primers by Sangon Biotech (Shanghai, China).
6
Quantifying Human Gene Expression
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RNA extraction and reverse transcription into cDNA were conducted as described above for mouse retina. Reactions containing 50 ng of cDNA were subjected to qRT-PCR using 2 × Rapid Taq Master Mix (P222, Vazyme Biotech, Nanjing, China), the human-specific primers described above, and an ABI2720 Thermal Cycler (Allied Biosystems). The thermocyle was 95 °C for 3 min, followed by 35 cycles of 95 °C for 15 s, 58 °C for 15 s, 72 °C for 15 s, and a final extension at 72 °C for 5 min. The PCR product was analyzed by 2% agarose gel electrophoresis.
The human GAPDH gene served as the internal control.
The human GAPDH gene served as the internal control.
7
Mitogenome Assembly of Trichogramma Species
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For T. cacoeciae, the full-length mitogenome was assembled by Geneious v9.1.4 [33 (link)] using Illumina reads and the Sanger-sequenced Cox1 sequence. For T. pretiosum, Illumina reads were first assembled using MitoZ v2.4 [34 (link)], and the Cox1 sequence was then merged into the assembly using Geneious v9.1.4. Since there was still a gap between Cox1 and Cox3, additional primers, FP4 and RP4, were designed using Primer Premier 5 to fill this gap (Figure 1 ; Table S1 ). We also noticed an erroneous insertion of 14 amino acids within Nad4l in the T. pretiosum mitogenome assembly. A pair of primers, FP5 and RP5, were designed for amplification and Sanger-sequencing of this insertion region (Figure 1 ; Table S1 ). This erroneous insertion was corrected by the Sanger-sequencing result. PCRs in this section were performed using Rapid Taq Master Mix (Vazyme, Nanjing, China). The PCR cycling settings were as follows: activation for 3 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 54 °C, and 1 min at 68 °C. The final cycle was followed by an extension of 10 min at 68 °C.
8
Mutation Detection in Grapevine Using Sanger and High-Throughput Sequencing
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To detect the mutations at the designed targets, the sequences containing the target sites were first amplified by PCR using site-specific primers from grape cells or grapevine plants. Mutations were then detected using Sanger sequencing or high-throughput sequencing. The mutations in grapevine plants were detected using Sanger sequencing. For Sanger sequencing assay, the amplified target fragments were ligated into pLB vector (TIANGEN), and a number of 6 clones were randomly picked and analyzed by Sanger sequencing. The mutations in 41B cells were detected using Hi-TOM assay (http://www.hi-tom.net/hi-tom/ ). For Hi-TOM assay, target-specific primers with bridging sequences at 5’ end were used to amplify the target fragments using the rapid Taq master mix (Vazyme) according to the manufacturer’s protocol. The PCR products were used for NGS after a second-round PCR called barcoding PCR. The mutations were determined by analyzing the sequencing reads according to the protocol described by Liu et al., (2019 (link)). All the primers are available in Supplementary Table S13 .
9
Quantification of VlhA gene by qPCR
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The lengths of the optimal primer pair MS F3/MS R3 were modified according to the requirements of PCR, F:5’-ATTACTATTAGCAGCTAGTGCA-3’, R:5’-ACCTGGATTTCCTGGAGTACCTGG-3’. The modified primers were used to amplify the DNA fragment by a PCR kit (Vazyme Biotech Co., Ltd., Nanjing, China) with the following reaction system (50 μL): 2 × Rapid Taq Master Mix, 25 μL; forward (10 μM) and reverse (10 μM) primers, 2 μL each; DNA template, 1 μL; sterile water, 20 μL. The DNA extracted from MS strain GX11-T was used as the positive control. The PCR procedure consisted of a pre-denaturation at 95°C for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s and extension at 72°C for 10 s; then a final extension at 72° for 5 min. The amplified product was purified with the DNA fragments purification kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) and ligated to pUCm-T Vector (Beyotime Biotechnology Co., Ltd., Shanghai, China). The resulting plasmid pUCm-VlhA was quantified with a spectrophotometer at 260 nm, and its copy number was calculated as the following formula: plasmid copy number (copies/μL) = [plasmid concentration (g/μL) × 6.02 × 1023/plasmid length (bp) × 660 g/mol]. Ten-fold dilutions of the plasmid pUCm-VlhA with the gradient ranging from 107 to 100 copies/μL were prepared and stored at −20°C.
10
Verification of Chromosome Modifications
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To verify the elimination of BY4741XT1-FCY1 and BY4742XT2-URA3 chromosomes, we used the junctions between FCY1 or URA3 and chromosomes at the BY4741XT1-FCY1 or BY4742XT2-URA3 centromere as specific PCRTags by colony PCR to distinguish the chromosome-modified and WT strains. Briefly, a single yeast colony was resuspended in 10–40 µL of 20 mM NaOH and placed in the thermocycler. Yeast colony suspensions were boiled at 95 °C for 5 min and then cooled at 4 °C for at least 5 min before PCR was performed. 1 µL of yeast lysate was used as a template in a 10 µL 2× Rapid Taq Master Mix (Vazyme P222-AA) with 0.25 µM of primers. PCR program: 95 °C, 5 min; 30× (95 °C, 20 s; 55 °C, 90 s; 72 °C, 1 min); 72 °C, 5 min; 4 °C, hold. PCR products were separated on a 1% agarose gel containing ethidium bromide. 2 kb Plus II DNA Ladder (TransGen BM121-01) was used as a molecular weight standard.
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