Bandage v0.8.1 [64 (link)] was used to merge contigs with pairwise connections and generate a single connected sequence, based on the mitogenome conformation resolved for IS929. Then Primer Premier 6 (Premier Biosoft Interpairs, Palo Alto, CA) was used to design primers in the range of 0.5–2 kbp on both sides of each linkage site for each linkage variant. The DNA isolated from young leaf tissue of S. bicolor ssp. bicolor (IS929) was used to conduct PCR verification. PCR amplification products that crossed linkage sites were then used to verify each linkage relationship (Suppl. Table 2 ). PCRs were performed in volumes of 20 uL consisting of 1 uL template DNA, 0.4 uL 2.5 mM dNTP, 2 uL 10 × EasyTaq® Buffer (TransGen), 0.2 uL 500U EasyTaq® DNA Polymerase (TransGen), 0.2uL 100uM forward primer, 0.2uL 100uM reverse primer, and 16 uL ddH2O. Thermocycling conditions were 95℃ denaturation for three minutes, followed by 35 cycles each including 95℃ denaturation for 30 s, 60 ~ 61.5 ℃ annealing for 30 s (60 ℃ for ctg9-8; 60.5 ℃ for ctg1-5, 2–4, 3–5, 4–1, 6–3, 6–7, 7–4, 8–6, 9–4, 9–5; 61.5 ℃ for ctg6-2), and 72℃ extension for three minutes. Following the 35 cycles a final 5-min extension step at 72℃ was conducted. The PCR products were assessed for length using a 1.5% agarose gel run at 100 V for 25 min and compared to an 8 kbp ladder.
Easytaq dna polymerase
Manufactured by Transgene
Sourced in China, United States
EasyTaq DNA polymerase is a thermostable DNA polymerase used for DNA amplification in PCR applications. It possesses 5'→3' DNA polymerase and 3'→5' exonuclease activities.
Automatically generated - may contain errors
Lab products found in correlation
Easytaq buffer, by Transgene (10 mentions)
Dntps, by Transgene (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (3 mentions)
Tb green premix ex taq 2, by Takara Bio (3 mentions)
Antibody for nf κb, by Cell Signaling Technology (3 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
100 bp plus 2 dna ladder, by Transgene (3 mentions)
Picogreen, by Thermo Fisher Scientific (2 mentions)
Nextera xt index kit, by Illumina (2 mentions)
Miseq sequencer, by Illumina (2 mentions)
16s metagenomic sequencing library preparation, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Sodium thioglycolate, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Merck Group (2 mentions)
Mycycler thermal cycler, by Bio-Rad (2 mentions)
Easytaq dna polymerase buffer, by Transgene (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
69 protocols using easytaq dna polymerase
1
Mitogenome Assembly and Validation
2
Primer Design and PCR Optimization
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The selected loci were amplified by new primers designed using the primer design tool in Geneious R11 (Supplementary Table 6 ). PCRs were carried out using EasyTaq DNA Polymerase (Transgen Biotech, Beijing, China) following the manufacturer’s manual. A standard thermocycling profile was applied, and the annealing temperature was successful at 55 °C for all primers. Amplicons were revealed using EtBr-stained 1.5% agarose gel electrophoresis under UV light. For loci that accumulated high interspecies/variety SNPs, when successful, the PCR product was subjected to purification and sequencing (Tsingke, Wuhan, China).
3
Bacterial 16S rRNA Phylogenetic Analysis
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Bacterial genomic DNA was extracted by the phenol/chloroform method [18 ]. The 16S ribosomal DNA (rRNA) was amplified from extracted DNA by polymerase chain reaction with primers 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-ACGGTTACCTTGTTACGACTT-3′ [19 (link)]. Amplification was performed by initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 51 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The 50-μL PCR mixtures contained 0.2 μM of each primer, 0.2 mM dNTPs, EasyTaq® buffer, 2.5 U EasyTaq® DNA polymerase (TRANSGEN, China) and 10 ng of template DNA. The PCR products were sequenced by Beijing Sun Biotech Co., Ltd. (Beijing, China). The sequences were then aligned to reference 16S rRNA sequences in the NCBI database using the BLAST program with default parameters [20 ]. The sequenced 16S rRNAs and reference 16S rRNAs were subjected to phylogenetic analysis using MEGA7 software [21 (link)]. The sequences were aligned by ClustalW to reconstruct a phylogenetic tree using the Kimura 2-parameter distance model and neighbor-joining method (1000 bootstrap replicates).
4
Quantitative PCR and RT-qPCR protocols for gene expression analysis
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PCR reactions were performed using a standard thermocycler (Bio-rad T100, Hercules, CA, USA), according to the manufacturer’s protocols. Each 25 μL of PCR reaction contained 2.5–5 U EasyTaq DNA polymerase (Transgen Biotech, Beijing, China), 10× Ex Taq buffer, 0.2 mM dNTPs (2.5 mM), 0.2 μM of each primer, and 1 ng-1μg template DNA. PCR conditions were 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 56–60 °C, 1–2 kb/min at 72 °C, and 10 min at 72 °C. Primer sequences are listed in Table S1 .
RT-qPCR was performed with a CFX96 Real-Time PCR Detection System (Analytik Jena, Germany), according to the manufacturer’s protocols. Each 25 μL of PCR reaction contained 5 μL of ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China), 1 ng-100 ng cDNA, and 0.2 μM of each primer. The reaction procedure was 95 °C 30 s, followed by 40 cycles of 5 s at 95 °C, 30 s at 60 °C; melt curve. Samples collected at 0 hpi were used to calibrate expression levels. The β-actin gene of P. italicum was used as an internal reference [17 (link)]. A combination of three biological replicates with three technical replicates of each reaction were used. The relative expression levels of genes were calculated by the 2−ΔΔCt method [38 (link)].
RT-qPCR was performed with a CFX96 Real-Time PCR Detection System (Analytik Jena, Germany), according to the manufacturer’s protocols. Each 25 μL of PCR reaction contained 5 μL of ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China), 1 ng-100 ng cDNA, and 0.2 μM of each primer. The reaction procedure was 95 °C 30 s, followed by 40 cycles of 5 s at 95 °C, 30 s at 60 °C; melt curve. Samples collected at 0 hpi were used to calibrate expression levels. The β-actin gene of P. italicum was used as an internal reference [17 (link)]. A combination of three biological replicates with three technical replicates of each reaction were used. The relative expression levels of genes were calculated by the 2−ΔΔCt method [38 (link)].
5
High-Resolution Melting Analysis of DNA
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The total reaction volume for HRM analysis was 20 μl, containing 10 ng of genomic DNA, 2.0 μl of 10 × Easy Taq buffer (TransGen Biotech, Beijing, China), 1.0 μl of 2.5 mM dNTP mixture (TransGen Biotech), 0.1 μl of Easy Taq DNA polymerase (Transgen Biotech), 1.0 μl of SYTO®9 green fluorescent nucleic acid stain (Life Technologies™, Carlsbad, CA, United States), 0.5 μl each of 10 pmol μl−1 of a pair of primers (Supplementary Table S1 ), and autoclaved distilled water for the remainder of the volume. On the Biometra TAdvanced (Biometra GmbH, Göttingen, Germany), the following PCR conditions were used: initial denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, followed by annealing and elongation at 60°C for 20 s, repeated 40 times; and final denaturation at 95°C for 10 s. HRM analysis was performed using the LightCycler® 96 Real-Time PCR System (Roche, Basel, Switzerland) at each temperature during a 0.3% rise from 60 to 90°C. The HRM graphs were drawn by LightCycler® 96 software ver. 1.1 (Roche).
6
Identifying TERT Promoter Mutations
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Mutations causing altered TERT expression were studied using NGS, following the 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA) protocol. To identify TPMs (chr5:1,295,228 C > T and chr5: 1,295,250 C > T), amplicons of 151bp were amplified from 50 ng of tumor DNA (primer sequences provided in Table S2 ). Amplicon PCR was performed using Multiplex QIAGEN 2X Master Mix following manufacturer’s instructions. Index PCR was later executed with the EasyTaq DNA polymerase (TransGen Biotech, Beijing, China) using synthetic indices from the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Both PCR products were purified using AMPure XP beads (Beckman Coulter, Pasadena, IN, USA) and quantified by PicoGreen (Thermo Fisher Scientific, Waltham, MA, USA). Amplicon concentration was normalized and pooled up to 96 samples for a single run.
Libraries were sequenced according to manufacturer’s instructions in a MiSeq sequencer (Illumina). The sequencing module used was the “PCR Amplicon” protocol with a paired-end design with 150 base pairs reads. Illumina software was used to perform the variant calling, and Illumina VariantStudio software (Illumina) was used to obtain the sequencing results. TERT promoter mutations detected by NGS were confirmed by PCR and Sanger sequencing (primers are provided inTable S2 ).
Libraries were sequenced according to manufacturer’s instructions in a MiSeq sequencer (Illumina). The sequencing module used was the “PCR Amplicon” protocol with a paired-end design with 150 base pairs reads. Illumina software was used to perform the variant calling, and Illumina VariantStudio software (Illumina) was used to obtain the sequencing results. TERT promoter mutations detected by NGS were confirmed by PCR and Sanger sequencing (primers are provided in
7
Mouse Tail DNA Extraction and Genotyping
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Total DNA was extracted from the mouse tail by using the EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China), and the genotype was screened using PCR (EasyTaq DNA Polymerase, Transgen Biotech, Beijing, China). The primers are listed in Table 1 .
8
Microbial Immunology and Molecular Characterization
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Sodium thioglycolate, 4′,6-diamidino-2-phenylindole dilactate (DAPI), LPS, and RPMI 1640 were purchased from Sigma (St Louis, MO). A De Man, Rogosa, and Sharpe (MRS) medium, Sabouraud dextrose agar (SDA), and general anaerobic medium were purchased from BD (Franklin Lakes, NJ). An antibody for NF-κB was purchased from Cell Signaling Technology (Danvers, MA). Antibodies for CD11c, Iba1, and BDNF were purchased from Abcam (Cambridge, U.K.). Alexa Fluor 488 and Alexa Fluor 594 were purchased from Invitrogen (Carbsband, CA). Enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ, TNF-α, and IL-10 were purchased from Ebioscience (Atlanta, GA). A QIAamp Fast DNA stool mini kit was purchased from Qiagen (Hilden, Germany). EasyTaq DNA polymerase and 100 bp plus II DNA ladder were purchased from TransGen Biotech (Beijing, China). TB Green® Premix Ex Taq™ II was purchased from Takara Bio (Shiga, Japan).
9
PCR Amplification and Sequencing Protocol
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PCR was performed in a total volume of 25 μL containing 2.5 μL of 10× EasyTaq® buffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of forward and reverse primers (10 μM), 0.25μL of EasyTaq® DNA Polymerase (Transgen Biotech Co., Ltd., Beijing, China), and 1 μL of genomic DNA templates. The thermocycling parameters were as follows: initial denaturation at 94 °C for 5 min, followed by 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s for 35 cycles, completed with a final extension at 72 °C for 7 min. The PCR products were sent to General Biol Co., Ltd. (Anhui, China) for sequencing. The obtained sequences were edited for further analysis.
10
Fungal Genomic DNA Extraction and Characterization
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Fungal genomic DNA was extracted as described previously (10 (link)). Plasmids and PCR products were purified using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, Norcross, GA). Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, Massachusetts) and EasyTaq DNA polymerase (TransGen Biotech, Beijing, China) were used for PCRs. Sequences were determined by SinoGenoMax Co., Ltd. (Beijing, China). Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs (Ipswich, Massachusetts).
RNA was extracted with the Qiagen RNeasy minikit, and carryover DNA was removed by DNase I digestion (Qiagen, Valencia, CA). cDNA fragments were synthesized using the TransScript II first-strand cDNA synthesis supermix (TransGen Biotech, Beijing, China). Introns were identified by comparing genomic and cDNA sequences. The 5′ ends of the mRNA and the poly(A) attachment sites were mapped by 5′- and 3′-RACE–PCR (FirstChoice RLM-RACE kit; Ambion, Austin, Texas).
All primers used in the study are listed inTable S3 in the supplemental material.
RNA was extracted with the Qiagen RNeasy minikit, and carryover DNA was removed by DNase I digestion (Qiagen, Valencia, CA). cDNA fragments were synthesized using the TransScript II first-strand cDNA synthesis supermix (TransGen Biotech, Beijing, China). Introns were identified by comparing genomic and cDNA sequences. The 5′ ends of the mRNA and the poly(A) attachment sites were mapped by 5′- and 3′-RACE–PCR (FirstChoice RLM-RACE kit; Ambion, Austin, Texas).
All primers used in the study are listed in
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