The extracted DNA was subjected to polymerase chain reaction targeting A. ceylanicum and A. tubaeforme internal transcribed spacer 1 (ITS1) sequences using a forward primer AF (5′-CTTTGTCGGGAAGGTTGG-3′) and a reverse primer AR (5′-TTCACCACTCTAAGCGTCT-3′) previously designed by Liu et al. [4 (link)]. The target amplification fragment length was 404 bp. PCR was carried out in a final volume of 25 μl, containing 12.5 μl Premix Ex-Taq polymerase (TaKaRa, Dalian, China), 9.5 μl ddH2O, 0.5 μl of each primer AF/AR (50 μmol/L), and 2 μl DNA template. The utilized PCR program was as follows: 94°C for 5 min, 35×(94°C for 30 sec, 61.5°C for 30 sec and 72°C for 45 sec), and 72°C for 7 min. The PCR products were assessed by electrophoresis in agarose gel (1.5%) with ethidium bromide stain (0.2 mg/ml) and visualized on a UV transilluminator. The amplified fragment was recovered from the gel following the protocol of the gel recovery kit (Omega, Guangzhou, China) and kept at −20°C until further use.
Premix ex taq polymerase
Manufactured by Takara Bio
Sourced in China, Japan
Premix Ex-Taq polymerase is a ready-to-use PCR reaction mixture that contains Taq DNA polymerase, dNTPs, and buffer components. It is designed to provide a simple and efficient solution for PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Mini sub cell gt system, by Bio-Rad (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Fastking one step rt pcr kit, by Tiangen Biotech (1 mentions)
Qiaamp rna kit, by Qiagen (1 mentions)
Abi prism 7500 system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using premix ex taq polymerase
1
Molecular Detection of Hookworm Species
2
ITS1 Amplification of Ancylostoma Parasites
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The ITS1 sequences of A. ceylanicum and A. caninum were amplified using a forward primer AF (5′-CTTTGTCGGGAAGGTTGG-3′) and a reverse primer AR (5′-TTCACCACTCTAAGCGTCT-3′) designed by Liu et al. [15 (link)]. The predictive amplification fragment was 404 bp. Polymerase chain reactions were performed in 25 μL, including 12.5 μL of Premix Ex-Taq polymerase (TaKaRa, Dalian, China), 9.5 μL of ddH2O, 0.5 μL of each primer AF/AR (50 μmol/L), and 2 μL of DNA sample. PCR cycling parameters were as follows: 1 cycle at 94°C for 5 min and then 35 cycles at 94°C for 30 sec, at 61.5°C for 30 sec, and at 72°C for 45 sec, followed by 1 cycle at 72°C for 7 min. The PCR products were analysed by gel electrophoresis in 1.5% agarose gels, stained with 0.2 mg/ml ethidium bromide, and visualized on a UV transilluminator.
3
Detecting Nitrogen Cycling Genes in Soil
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Conventional PCR was performed to determine the existence of target sequences in DNA extracted from −99-h soil cores. A 25-μL reaction mixture contained 15 ng of soil DNA, primers (see Supplementary Table S1 ) at a final concentration of 0.2 μM (nirS and nirK) or 0.8 μM (nosZ), bovine serum albumin at a final concentrations of 0.48 mg mL−1 (nirS and nirK) or 1 mg mL−1 (nosZ), nuclease-free water, and the Premix Ex Taq polymerase (hot-start version; Takara Bio, Kusatsu, Shiga, Japan). Dimethyl sulfoxide was added to a final concentration of 5% to amplify cluster III sequences of nirK. A Veriti thermal cycler (Thermo Fisher Scientific) was used to perform conventional PCR. Cycling parameters for conventional PCR are listed in Supplementary Table S2 .
The genes detected by conventional PCR in DNA extracted from −99-h soil cores were subjected to qPCR analysis using a QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). A 1-μL sample of 2-fold diluted soil DNA was used as a template in a 20-μL reaction mixture. The final concentration of each primer in the PCR mixture was 1 μM. Cycling parameters are listed in Supplementary TableS3 .
The genes detected by conventional PCR in DNA extracted from −99-h soil cores were subjected to qPCR analysis using a QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). A 1-μL sample of 2-fold diluted soil DNA was used as a template in a 20-μL reaction mixture. The final concentration of each primer in the PCR mixture was 1 μM. Cycling parameters are listed in Supplementary Table
4
RT-PCR Splicing Analysis of VviDXS and VviCRTISO
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The reverse transcription polymerase chain reaction (RT-PCR) splicing analysis of VviDXS and VviCRTISO was performed in a 10 μL reaction volume. Each reaction contains 1 μL of the cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 3 μL ddH2O, and 5 μL of 2 × Premix Ex-Taq polymerase (Takara). The cycling conditions were 98 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a 60 s extension at 72 °C, with a final 10 min extension at 72 °C. The PCR amplicons were analyzed using 2% agarose gel electrophoresis. The primers were designed from the upstream and downstream exons of the skipped exon or retention intron (Table S9 ).
5
Comprehensive Gene Expression Analysis
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The sequences and corresponding PCR information for the primers used in this study are listed in S1 Table . Premix Ex Taq polymerase (Takara D332) was used for PCRs. Trizol (Invitrogen; Carlsbad, CA, USA) was used for RNA extraction and purification. The PrimeScriptTM RT kit (Takara RR047A) was used to perform the reverse transcription reactions. A T100 Thermal Cycler (Bio-Rad; Hercules, CA, USA) was used for PCR amplification. DNA electrophoresis was performed using a Mini-Sub cell GT system and a Gel imaging system (Bio-Rad). All oligonucleotides used in this study are listed in S1 Table .
6
Orthohantavirus RNA Extraction and RT-PCR Detection
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Orthohantavirus nucleic acids were extracted from host animal liver and lung tissues as well as from patient blood samples using the QIAamp RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The viral RNA is stored in a −80 °C freezer until use. RT-PCR amplification of the L-gene fragment of orthohantavirus was performed on the extracted RNA with the orthohantavirus universal primer: HAN-L-F1: 5′-ATGTAYGTBAGTGCWGATGC-3′, HAN-L-R1: 5′-AACCADTCWGTYCCRTCAT-C-3′; HAN-L-F2: 5′-TGCWGATGCHACIAARTGGTC-3′, and HAN-L-R2: 5′-GCRTCRTCWGARTGRTGDGCAA-3′ [24 (link)], using the FastKing One-Step RT-PCR Kit (TIANGEN BIOTECH, Beijing, China) and Premix Ex Taq polymerase (TaKaRa Bio Inc, Shiga, Japan) according to the manufacturer’s instructions. Nested RT-PCR was performed with a total reaction system of 25 μL. Reaction conditions: reverse transcription at 42 °C for 30 min, pre-denaturation at 95 °C for 3 min, followed by denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min for 35 cycles, followed by supplemental extension at 72 °C for 30 s, and finally cooling at 12 °C for 1 min to be used. PCR products were extracted on a 1.2% agarose gel for electrophoretic analysis and observed in a UV gel imager for the presence or absence of target bands of specific molecular weight size (about 412 bp), and gel images were saved.
7
Gene Expression Analysis in MC3T3-E1 Cells
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Subconfluent MC3T3-E1 cells were treated as described for the ALP activity assay. Following treatment, cell total RNA was extracted with TRIzol (Thermo Fisher Scientific, Waltham, Massachusetts). Synthesis of complementary DNA (cDNA) was performed using the high-capacity cDNA reverse transcription kit following the manufacturer’s instructions (Applied Biosystems, Grand Island, New York). A quantitative real-time PCR was performed in an ABI PRISM 7500 system (Applied Biosystems). TaqMan minor groove binder probes (Applied Biosystems, Assay-by-Design) were obtained for amplification of the following genes: runt-related transcription factor 2 (Runx2), osterix (Osx) and ALP using Premix ex-Taq polymerase (Takara Bio Inc., Otsu, Japan). The messenger RNA (mRNA) copy numbers were calculated for each sample using the cycle threshold value and normalized against 18S ribosomal RNA, as previously reported.44 (link),45 (link) Results were expressed as n-fold mRNA values versus corresponding values in nontreated control cells.
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