Platinum taq dna polymerase

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Platinum Taq DNA polymerase is a thermostable DNA polymerase enzyme designed for high-performance PCR amplification. It exhibits robust activity and fidelity across a wide range of templates and applications.

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DNA platinum Taq DNA polymerase Invitrogen Platinum Taq DNA polymerase SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Superscript-III kit Platinum Taq DNA polymerase enzyme SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Platinum Taq DNA Polymerase High Fidelity Kit Platinum™ Taq DNA Polymerase system Platinum Taq DNA Polymerase High Fidelity SuperScript® One-Step RT-PCR System with Platinum® Taq DNA Polymerase Platinum Taq DNA Polymerase High Fidelity enzyme

869 protocols using platinum taq dna polymerase

Comprehensive Genetic Analysis of TAPBP

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HLA class I genotypes were determined by either the PCR–sequence-based typing protocol, as recommended by the 13th International Histocompatibility Workshop or by targeted next-generation sequencing, as described (11 (link)).
DNA from individuals in the three South African cohorts and the NCI RDP (the majority of the NCI RDP are of European descent) was sequenced for the 5′ UTR, exons, and 3′ UTR of TAPBP. Three gene segments were amplified with Platinum Taq DNA polymerase (Invitrogen) in several fragments: from the 5′ UTR to the beginning of the third intron (approx. 1.5 kb), from the end of the third intron to the beginning of the seventh intron (approx. 1.5 kb), and from the end of the seventh intron to the end of the 3′ UTR (approx. 2.1 kb) (see SI Appendix, Table S7, for primer sequences). The amplified fragments were sequenced by using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and run on a 3730 Genetic Analyzer (Applied Biosystems) (primers used for sequencing are listed in the SI Appendix, Table S7). rs111686073 and rs59097151 were genotyped in the individuals from the Ugandan cohorts by amplifying the two regions containing the SNPs with Platinum Taq DNA polymerase (Invitrogen) (primers used for amplification and sequencing are listed in the SI Appendix, Table S7).

Walker-Sperling V., Digitale J.C., Viard M., Martin M.P., Bashirova A., Yuki Y., Ramsuran V., Kulkarni S., Naranbhai V., Li H., Anderson S.K., Yum L., Clifford R., Kibuuka H., Ake J., Thomas R., Rowland-Jones S., Rek J., Arinaitwe E., Kamya M., Rodriguez-Barraquer I., Feeney M.E, & Carrington M. (2022). Genetic variation that determines TAPBP expression levels associates with the course of malaria in an HLA allotype-dependent manner. Proceedings of the National Academy of Sciences of the United States of America, 119(29), e2205498119.

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Multiplex qPCR for H. pylori and CYP2C19

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Five primers and nine matching Taqman probes targeting the H. pylori cagH and 23S rRNA genes and the human RnaseP, CYP2C19*2 and CYP2C19*3 genes were designed for this assay. All sequences obtained from NCBI Entrez Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore) were aligned using Vector NTI alignment software (http://www.lifetechnologies.com/cn/zh/home/life-science/cloning/vector-nti-software.html). The primers and probes were designed using Primer Express 3.0 software (Applied Biosystems). The sequences of the primers and probes used in this study are summarized in table 2.
The reaction mixture (20 μL) was prepared as follows: 2 μL 10× PCR buffer, 2 mM MgCl₂(Platinum® Taq DNA Polymerase, Invitrogen, Thermo Fisher, USA), 0.4 μL dNTPs (Promega, USA), 0.5 μM forward and reverse primers (Sangon Biotech, Shanghai, China), 0.2 μM probe (ABI, USA), 0.2 μL Taq DNA polymerase (Platinum® Taq DNA Polymerase, Invitrogen, Thermo Fisher, USA), 2 μL DNA template, and up to 20 μL nuclease-free water.

Peng X., Song Z., He L., Lin S., Gong Y., Sun L., Zhao F., Gu Y., You Y., Zhou L, & Zhang J. (2017). Gastric Juice-Based Real-Time PCR for Tailored Helicobacter Pylori Treatment: A Practical Approach. International Journal of Medical Sciences, 14(6), 595-601.

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Preparation of Biotinylated and Fluorescent DNA Templates

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Upstream biotinylated and downstream AF488 labeled DNA templates (Figure 1A) were prepared by PCR from pUC18-G5CYC1 G- (SB649; Table S2) with Platinum Taq DNA polymerase (Invitrogen) and primers Biotin-universal and AF488-G-less-PL_REV for the UAS and AF488-Mid G-less d2 REV(+17) for the UAS+promoter templates (primer sequence information in Table S3). The DNA templates used to test effects of the core promoters and 4NTPs were prepared by PCR from pUC18-G5CYC1 G- (SB649; Table S2) and pUC18-G5-HIS4 G- (SB1964; Table S2) for the CYC1 and HIS4 core promoter, respectively with Platinum Taq DNA polymerase (Invitrogen) and primers Biotin-universal and AF488-M13 rev2 (primer sequence information in Table S3). The PCR product was purified using DNA SizeSelector-I SPRI magnetic beads (Aline Biosciences). The DNA template sequence information is in Table S5.

Baek I., Friedman L.J., Gelles J, & Buratowski S. (2021). Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes. Molecular cell, 81(17), 3576-3588.e6.

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Quantification and Validation of circRNAs

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The expression of the five circRNAs was evaluated by RT-PCR, using divergent primers. Samples were analyzed in technical triplicates and biological quadruplicates. The Polymerase Chain Reactions (PCR) reactions were realized using the Platinum Taq DNA polymerase (Invitrogen) enzyme. All RT-PCR reactions were performed on the Applied Biosystems Veriti apparatus. PCR conditions were conducted in a volume of 20 μL containing 10 μL of the diluted cDNA (1:100), 0.4 mM dNTPs, 10× Buffer, 3 mM MgCl 2, 0.25 U Platinum Taq DNA polymerase (Invitrogen) and 0.1 μM of each oligonucleotide. PCR conditions were: an initial 2 min step at 95 °C followed by 40 cycles of 10 s denaturing at 95 °C, 15 s annealing at 60 °C and 15 s extension at 72 °C. Confirmation of the fragments was performed by 3% agarose gel electrophoresis. The circRNAs predicted by bioinformatics and confirmed by PCR were purified using the Wizard SV gel PCR clean-up system (Ludwig Biotecnologia) according to the manufacturer’s recommendations. Sanger sequencing reactions were performed with purified PCR products at a final concentration of the reaction of 4.5 pmol/μL, using the PcF, PuR, or PuRi primers (Supplementary Table S1).

Frydrych Capelari É., da Fonseca G.C., Guzman F, & Margis R. (2019). Circular and Micro RNAs from Arabidopsis thaliana Flowers Are Simultaneously Isolated from AGO-IP Libraries. Plants, 8(9), 302.

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RT-PCR Analysis of Gene Co-transcription

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Co-transcription was analysed by RT-PCR. RNA obtained at 48 h sucrose-free R5A liquid cultures. Reverse transcription was performed using the SuperScript one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen), using 200 ng of total RNA as template. Chromosomal DNA was used as template in the positive controls. RNA and the DNA polymerase included in the Platinum Taq DNA polymerase (Invitrogen) kit were used in the negative controls. RT-PCR was performed using the primers indicated in Table 4 (outlined in Fig. 1A) as follows. The first-strand complementary DNA (cDNA) synthesis was performed at 55 °C for 30 min followed by an initial denaturation at 94 °C for 2 min. Then, a touchdown was performed in 8 cycles during which the annealing temperature was reduced by 1 °C in each cycle: 94 °C for 15 s, 65 °C (Δ−1 °C) for 30 s and 68 °C for 38 s. For the next 35 cycles (94 °C for 15 s, 55 or 58 °C for 30 s and 68 °C for 38 s), the annealing temperature was set at 55 °C (for primers RT2829F/R) and 58 °C (for primers RT2930F/R, RT3031F/R and RT3031F1/R1). A final extension step was performed at 68 °C for 5 min.

González-Quiñónez N., Corte-Rodríguez M., Álvarez-Fernández-García R., Rioseras B., López-García M.T., Fernández-García G., Montes-Bayón M., Manteca A, & Yagüe P. (2019). Cytosolic copper is a major modulator of germination, development and secondary metabolism in Streptomyces coelicolor. Scientific Reports, 9, 4214.

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Quantitative Real-Time PCR Analysis of Gene Expression

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Cells were subjected to Trizol reagent (Life Technologies, Carlsbad, CA, USA) to extract total RNA. EcoDry TM Premix Oligo dT kits (Takara Bio, Shiga, Japan) were used for cDNA synthesis. The transcript level of the target gene was determined using TaqMan PreAmp Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The gene expression was normalized to GAPDH and was calculated using the delta-delta-Ct algorithm. Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA) was used to amplify the cDNA, subsequently used for PCR. Primers and probes are listed in Table S3. RT-PCR to verify pluripotency markers in iPSC clones was carried out using Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA). Primers for pluripotency markers were described in previous literature.³⁰ (link)

Rallabandi R., Sharp B., Cruz C., Wang Q., Locsin A., Driscoll C.B., Lee E., Nelson T, & Devaux P. (2021). miRNA-mediated control of exogenous OCT4 during mesenchymal-epithelial transition increases measles vector reprogramming efficiency. Molecular Therapy. Methods & Clinical Development, 24, 48-61.

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Quantitative Analysis of Transcript Splicing

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Retrotranscription was carried out with 400 ng of RNA and the RevertAid First Strand cDNA Synthesis Kit (Life Technologies), using the specific minigene primer RTPSPL3-RV (5′-TGAGGAGTGAATTGGTCGAA-3′). Samples were incubated at 42°C for 1 h, followed by 5 min at 70°C. Transcripts were amplified with Platinum Taq DNA polymerase (Life Technologies) using 40 ng of cDNA and the primers pMAD_607FW (Patent P201231427, CSIC) and RTBR2_ex17RV2 (5′-GGCTTAGGCATCTATTAGCA-3′). PCR consisted of: denaturation step at 94°C for 2 min, followed by 35 cycles 94°C-30 s, 60°C-30 s and 72°C-1 min/kb, and a final extension step at 72°C for 5 min. Transcripts were sequenced at the Macrogen Spain facility.
In order to relatively quantify all transcripts, semi-quantitative fluorescent RT-PCRs were undertaken in triplicate with the primers pMAD_607FW (FAM-labeled) and RTBR2_ex17RV2 and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied (Acedo et al., 2015 (link)). FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0 (Life Technologies). Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.

Fraile-Bethencourt E., Valenzuela-Palomo A., Díez-Gómez B., Caloca M.J., Gómez-Barrero S, & Velasco E.A. (2019). Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15. Frontiers in Genetics, 10, 503.

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Semiquantitative Fluorescent RT-PCR Analysis

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Retrotranscription was carried out with 400 ng of RNA and the RevertAid First Strand cDNA Synthesis Kit (Life Technologies), using the vector-specific primer RTPSPL3-RV (5'-TGAGGAGTGAATTGGTCGAA-3'). Samples were incubated at 42°C for 1 hour, followed by 5 min at 70ºC. Then, 40ng of cDNA (final volume of 50 µl) were amplified with SD6-PSPL3_RT-FW (5'-TCACCTGGACAACCTCAAAG-3') and RTpSAD-RV (Patent P201231427) (size 1,016 nt) using Platinum-Taq DNA polymerase (Life Technologies). Samples were denatured at 94ºC for 2 min, followed by 35 cycles of 94ºC/30 sec, 60ºC/30 sec, and 72ºC (1 min/kb), and a final extension step at 72ºC for 5 min. RT-PCR products were sequenced as previously indicated. Rare transcripts were subcloned into the pJet1.2 PCR cloning vector (Thermo Fisher Scientific) and sequenced.
In order to relatively quantify all transcripts, semiquantitative fluorescent RT-PCRs were undertaken in triplicate with primers PSPL3_RT-FW and RTpSAD-RV (FAM-labelled) and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied [20] . FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0. Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.

Fraile-Bethencourt E., Valenzuela-Palomo A., Díez-Gómez B., Goina E., Acedo A., Buratti E, & Velasco E.A. (2019). Mis-splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays. The Journal of pathology, 248(4).

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Nested PCR Amplification of nifH Gene

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Nested PCR amplification of the nifH gene was carried out in triplicate with the primers reported by Yeager et al. (2004 (link)). For the first reaction the nifH19F (5′-GCIWTYTAYGGIAARGGIGG-3′), and the nifH3R (5′-ATRTTRTTNGCNGCRTA-3′) were used. The reaction was performed in a 25-μl volume with the following reagent concentrations: 1X buffer, 2.5 mM MgCl₂, 2 mM (each) of dNTPs, 2 μM (each) of primers, 2.5 U of Taq DNA polymerase Platinum (Thermo Scientific), 10 ng of BSA, and 0.5 μl of DMSO and ∼ 40 ng of DNA. For the second reaction the primers nifH11F-FAM (fluorescently labelled; 5′-GAYCCNAARGCNGACTC-3′) and nifH22R (5′-ADWGCCATCATYTCRCC-3′). Reaction was performed in a 50-μl volume with the same reagent concentration as the previous reaction except for: 2.0 mM MgCl₂, 0.1 U of Taq DNA polymerase Platinum (Thermo Scientific), 2 μl of the first reaction, and without BSA and DMSO. Thermocycler parameters for the first reaction were as follows: 95°C for 5 min, followed of 20 cycles of 48°C for 1 min, 72°C for 1 min and 95°C for 45 s, with a 72°C final extension step for 10 min. For the second reaction, parameters were as described for the first reaction except that the annealing temperature was 55°C and the reaction cycle was repeated 32 times.

Barrón-Sandoval A., Martiny J.B., Pérez-Carbajal T., Bullock S.H., Leija A., Hernández G, & Escalante A.E. (2023). Functional significance of microbial diversity in arid soils: biological soil crusts and nitrogen fixation as a model system. FEMS Microbiology Ecology, 99(2), fiad009.

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Amplification and Sequencing of RNA-Protein Interactions

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PCR amplification was done with Platinum Taq DNA polymerase (Thermo Fisher Scientific 10966018) using 0.5 μM 3′ RT primer and 5′ SHORT PCR primer in a 100-μL reaction volume. After eight cycles from PCR (10 sec at 94°C, 30 sec at 60°C, and 15 sec at 72°C), the samples were concentrated and purified using DNA Clean & Concentrator-5 (Zymo Research, Catalog no. D4013). Products of a size between 75 and 100 bp were isolated using a 3% agarose PippinPrep cassette (Sage Science CSD3010) on a BluePippin device.
The second PCR was done with the purified and size-selected samples using Platinum Taq DNA polymerase together with 0.5 μM 5′ long PCR primer and 3′ RNA index primer (barcoded primer) in 100-μL reaction volume for 15 cycles. The PCRs were again purified, and size-selected for a range between 147 and 173 bp. The resulting library was sequenced on a HiSeq 3000 system using a single-end 50 cycle protocol. Analysis was performed as described previously using PARalyzer (version 1.5; Corcoran et al. 2011 (link)) built into the PARpipe (Corcoran et al. 2011 (link)) pipeline mapping the reads to human genome hg19. Pooled version of reads was mapped on human genome hg38.

Muys B.R., Anastasakis D.G., Claypool D., Pongor L., Li X.L., Grammatikakis I., Liu M., Wang X., Prasanth K.V., Aladjem M.I., Lal A, & Hafner M. (2021). The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma. Genes & Development, 35(1-2), 102-116.

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