Fifty microliter of PCR mastermix solution in Nuclease-free ultrapure water contained 60 mM Tris-HCl Buffer pH 8.8 (Bio-Rad), 200 µM dNTPs, 200 nmol µL−1 of each primer (Biomers), 0.5 U Taq DNA polymerase, and 20 µg mL−1 MB. Fifty microliter of PCR mix solution in Nuclease-free water contained 60 mM Tris-HCl Buffer pH 8.8 (Bio-Rad), 200 µM dNTPs, 200 nmol µL−1 of each primer (Biomers), 0.5 U Taq DNA polymerase, 2 mM MgCl2, 20 µg mL−1 MB, and 1 µL of MAP or Nuclease-free ultrapure water. Thirty microliter of PCR mastermix or PCR mix solutions were added into the sample reservoir of TriSilix for reaction, followed by 10 µL of mineral oil (Sigma) to prevent evaporation. All chemicals, unless otherwise stated, were purchased from Fisher Scientific.
Taq dna polymerase
Manufactured by Bio-Rad
Sourced in United States
Taq DNA polymerase is a thermostable enzyme derived from the thermophilic bacterium Thermus aquaticus. It is a DNA-dependent DNA polymerase that catalyzes the synthesis of new DNA strands complementary to a template DNA sequence, a core function required for various molecular biology techniques such as PCR (Polymerase Chain Reaction).
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Rt qpcr machine, by Bio-Rad (1 mentions)
Ptc 200 thermal cycler, by Bio-Rad (1 mentions)
Gel doc 2000 system, by Bio-Rad (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Mineral oil, by Merck Group (1 mentions)
Tris hcl buffer ph 8, by Bio-Rad (1 mentions)
Trisilix, by Merck Group (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
Gel red 10 000x, by Biotium (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Truseq stranded mrna sample prep kit, by Illumina (1 mentions)
Dnase set, by Qiagen (1 mentions)
Click it nascent rna capture kit, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
11 protocols using taq dna polymerase
1
Automated PCR Mix Preparation
2
Quantitative Gene Expression Analysis
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The mRNA expression of TNF-α, IL-1β, IL-6, E2F1, IκB, and p65 was evaluated by using RT-qPCR. For gene analysis, equal amounts of cDNA were added to a reaction mixture containing gene-specific forward and reverse primers deoxynucleotide Taq DNA polymerase and SYBR (Bio-Rad, Hercules, CA) in a reaction mixture. Quantification of cDNA was based on monitoring increased SYBR fluorescence during exponential phase amplification in an RT-qPCR machine (Bio-Rad, Hercules, CA), and the determination of the PCR cycle number at which the amplified product exceeded a defined threshold.
3
Genomic DNA Isolation and PCR Amplification
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Genomic DNA was isolated from 2 g fresh leaves of plants at three-leaf stage with SDS-phenol-chloroform method according to Sharp et al. [28 (link)] and Devos et al. [29 (link)] and purified to eliminate RNA, amylase and other unwanted compounds. The purity and concentration of DNA was assessed with microplate reader (M200, TECan, Switzerland). The DNA was finally diluted to concentration of 50 ng/μl and stored at −20 °C until use.
PCR amplification was carried out in a 10 μl reaction volume containing 40 ng genomic DNA, 2 μM each of the primer pairs, 2.5 mM each dNTPs, 2.5 mM MgCl2, 1 × PCR buffer (10 mM Tris–HCl, pH 8.5, 50 mM KCl), and 0.5 U Taq DNA polymerase with a PTC-200 thermal cycler (Bio-Rad, Hercules, CA, USA). The amplification was conducted an initial denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, annealing of different primers at 50, 55, or 60 °C for 50 s at a ramp rate of 0.5 °C/s, 72 °C for 1 min 10 s, and a final extension at 72 °C for 10 min. PCR products were resolved in 8% non-denaturing poly-acrylamide gels (Acr : Bis = 19 : 1 or 39 : 1) and the band patterns were visualized by silver staining [30 (link)].
PCR amplification was carried out in a 10 μl reaction volume containing 40 ng genomic DNA, 2 μM each of the primer pairs, 2.5 mM each dNTPs, 2.5 mM MgCl2, 1 × PCR buffer (10 mM Tris–HCl, pH 8.5, 50 mM KCl), and 0.5 U Taq DNA polymerase with a PTC-200 thermal cycler (Bio-Rad, Hercules, CA, USA). The amplification was conducted an initial denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, annealing of different primers at 50, 55, or 60 °C for 50 s at a ramp rate of 0.5 °C/s, 72 °C for 1 min 10 s, and a final extension at 72 °C for 10 min. PCR products were resolved in 8% non-denaturing poly-acrylamide gels (Acr : Bis = 19 : 1 or 39 : 1) and the band patterns were visualized by silver staining [30 (link)].
4
GC Tissue Sampling and Gene Expression Analysis
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Five matched GC tissue samples from patients who were diagnosed at the Department of Gastrointestinal Surgery of the First Affiliated Hospital of Wenzhou Medical University were obtained between October 2021 and January 2022. The distance between the tumor tissue and adjacent normal tissue was > 5 cm. This study received approval from the Institutional Ethics Committees of the First Affiliated Hospital of Wenzhou Medical University and followed the guidelines of the Declaration of Helsinki. The ethics ID number related to experiments in this study is 2019–089. All participants were fully informed and signed informed consent forms prior to participating in this research. Total RNAs from the tumor and adjacent tissues of GC patients were extracted using TRIzol reagent (Invitrogen). The cDNA synthesis was done via Taq DNA Polymerase (Bio-Rad) and the qRT-PCR was performed on the CFX96 optics module connected to a C1000 thermocycler system (Bio-Rad). β-actin was used as internal control, and fold change (2−△△CT) was used to express the relative gene expression. The primer sequences are listed in Supplementary Table S1 .
5
Identification of S. cerevisiae species
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For S. cerevisiae species identification, a set of pairs of species-specific primers ScerF2 (5′-GCGCTTTACATTCAGATCCCGAG-3′) and ScerR2 (5′-TAAGTTGGTTGTCAGCAAGATTG-3′) were used.12 (link) DNA amplification was carried out in a final volume of 25 μL containing 0.2 mM of dNTP (Invitek, Germany), 0.5 μL of each primer 100 pmol μL−1 (GeneriBiotech, Czech Republic), 1× PCR reaction buffer, 1.5 mM MgCl2 and 1.25 U Taq DNA polymerase (BioRad, USA) and 0.5 μL of template DNA. PCR cycling conditions used were: initial denaturation cycle at 94 °C for 4 min followed by 30 cycles of amplification, denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min; final extension at 72 °C for 2 min.
6
Total RNA Extraction and RT-PCR Analysis
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Total RNA was isolated using Trizol reagent (Invitrogen) as per the manufacturer’s recommendation. One microgram of total RNA was used as template for cDNA synthesis with cDNA synthesis kit (Bio-Rad Laboratories, USA) following the manufacturer's instructions.
RT-PCR analysis using primers listed in supplementary table 1 was performed by following the method described earlier (Pandey and Pandey-Rai 2014 ). All reactions were run in triplicate along with a negative control. Primer accuracy was tested using gDNA as positive control. PCR products were run on agarose gel and band intensities were measured using Quantity One software (Bio-Rad) installed with Gel Doc 2000 system (Bio-Rad).
PCR was performed in reaction mixture (50 µl final volume) containing 50 ng cDNA, 2.5 µl 10× PCR buffer with 15 mM MgCl2, 200 µM dNTPs, 10 pmol of each primer and 0.2 U Taq DNA polymerase (Bangalore Genei, India) in an iCycler (Bio-Rad, USA). The thermal amplification protocol was as follows: 1 cycle at 95 °C for 5 min, and 30 cycles at 95 °C for 30 s, 59 °C (value varied for different primer sets) for 45 s and 72 °C for 45 s.
RT-PCR analysis using primers listed in supplementary table 1 was performed by following the method described earlier (Pandey and Pandey-Rai 2014 ). All reactions were run in triplicate along with a negative control. Primer accuracy was tested using gDNA as positive control. PCR products were run on agarose gel and band intensities were measured using Quantity One software (Bio-Rad) installed with Gel Doc 2000 system (Bio-Rad).
PCR was performed in reaction mixture (50 µl final volume) containing 50 ng cDNA, 2.5 µl 10× PCR buffer with 15 mM MgCl2, 200 µM dNTPs, 10 pmol of each primer and 0.2 U Taq DNA polymerase (Bangalore Genei, India) in an iCycler (Bio-Rad, USA). The thermal amplification protocol was as follows: 1 cycle at 95 °C for 5 min, and 30 cycles at 95 °C for 30 s, 59 °C (value varied for different primer sets) for 45 s and 72 °C for 45 s.
7
Bacterial 16S rRNA and pheS Gene Amplification
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Total nucleic acids were extracted from each isolate using a miniprep protocol (Birnboim & Doly, 1979 (link)). A 1.5 kb fragment of the 16S rRNA gene was amplified using the primer pair 27F/1492R (Edwards et al., 1989 (link)). The PCR was done considering the conditions of an initial denaturation step at 94 °C for 1 min, 30 cycles of 94 °C for 40 s, 55 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. The master mix contained a final volume of 25 µl and included 1X reaction buffer, 0.2 mM dNTPs, 0.2 µM of each primer, 1.5 mM MgCl2, 1 U Taq DNA polymerase (Bio-Rad, Hercules, CA, USA), and 50 ng of DNA. In addition, a ∼490 bp fragment of the phenylalanyl-tRNA synthase (pheS) gene was amplified by PCR using the primer pair combination pheS-21-F/pheS-22-R (Naser et al., 2005 (link)). The reaction was performed using iProof High-Fidelity DNA polymerase (Bio-Rad) and 50 ng of DNA. The following cycling conditions were used: 98 °C for 30 s, 35 cycles of 98 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 10 min. PCR products were visualized by electrophoresis in a 1% agarose gel and stained with GelRed (10.000 X) (Biotium, Fremont, CA, USA). The amplified gene fragments were sequenced in both orientations by Macrogen® (Seoul, South Korea).
8
RNA Extraction, Sequencing, and Analysis
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Total RNA was purified with RNeasy Mini kit with DNase set (Qiagen). For RT-qPCR, cDNA was synthesized with ReverTra Ace qPCR RT Master Mix (TOYOBO). cDNA fragments were quantified by a SYBR green qPCR with gene specific primers and Taq DNA polymerase (NEB) with CFX96 Touch real-time PCR detection system (Bio-Rad). For Nascent RNA analysis, 1 × 106 cells were treated with 500 nM dTAG-13 for 1 h or 24h. 500 nM EU was added and incubated for additional 0.5 h. After the EU incorporation, total RNA was extracted with Direct-zol RNA miniprep kit (Zymo Research) at 1.5 h or 24.5 h post-dTAG-13 treatment. RiboMinus Human/Mouse Transcriptome Isolation Kit (ThermoFisher) was used for the removal of rRNA, and then EU-labelled RNA was enriched by using Click-iT Nascent RNA Capture kit (ThermoFisher). Libraries for RNA-seq were prepared by using the TruSeq Stranded mRNA Sample Prep Kit (Illumina). The DNA library was validated using TapeStation (Agilent Technologies) and was quantified using a QuantiFluor dsDNA system (Promega) and a Quantus Fluorometer (Promega). Libraries were pooled and sequenced on Illumina HiSeq 1500 or NextSeq 500. Data were analyzed by using HISAT2 and Cufflinks, or workflows on Basepair (https://www.basepairtech.com ). List of interest genes were analyzed by Enrichr tool (https://amp.pharm.mssm.edu/Enrichr/ ).
9
Identifying Quinolone Resistance Determinants
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To identify the QRDR mutations in the mutant strains, gyrA, gyrB, parC and parE were amplified and sequenced using the primers listed in Table S2 [18 (link)]. Some primers were constructed using A. pleuropneumoniae S4074 gene sequence data (GenBank accession number NZ_CP030753.1). PCR was performed in 50-μL volumes consisting of 2.5 U of TaKaRa Taq DNA polymerase, 200 μM of dNTPs, 1.5 mM MgCl2 and 50 pmol of each primer in a Bio-Rad T100TM Thermal Cycler for 33 cycles of denaturation at 94 °C for 30 s, annealing at 55–60 °C for 30 s and extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. PCR-amplified products were purified with the QIAquick PCR Purification Kit, following the manufacturer’s recommendations for sequencing. Sequencing was performed using an ABI prism 3730 Automated DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence analysis was carried out by multiple sequence alignment using DNAMAN and MEGA-X. Homology models were created using SWISS-MODEL [50 (link)].
10
Screening Virulence Genes in Isolates
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All 80 isolates were screened for the presence of virulence genes by PCR. Briefly, PCR was performed in a 30 μL reaction consisting of 22.1 μL of ultra-pure water, 3 μL of assay buffer (10X) (100 mM Tris-HCL (pH 9), 1.5 mM MgCl2, 50 mM KCL and 1% gelatin), 0.6 μL of four dNTP mix (200 mM), forward and backward primers (10 pmol of each) (Table S1 ), Taq DNA polymerase (1.0 U) and 2 μL of template DNA in a thermal cycler (Bio-Rad, USA).
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