Pcr advantage kit

Manufactured by Takara Bio

Sourced in Germany

The PCR Advantage Kit is a comprehensive solution for performing polymerase chain reaction (PCR) experiments. It includes all the necessary components, such as a thermostable DNA polymerase, reaction buffers, and PCR primers, to amplify target DNA sequences. The kit provides a streamlined and efficient way to conduct PCR-based studies.

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Lab products found in correlation

Spin miniprep kit, by Qiagen (3 mentions) Smarter race cdna amplification kit, by Takara Bio (3 mentions) Top10 competent cell, by Thermo Fisher Scientific (3 mentions) Rnaeasy mini kit, by Qiagen (2 mentions) Dna 12000 kit, by Agilent Technologies (1 mentions) Primescript reverse transcriptase, by Takara Bio (1 mentions) Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions) Monarch dna gel extraction kit, by New England Biolabs (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions)

5 protocols using pcr advantage kit

Long-range PCR and PacBio Sequencing of BCR-ABL1

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Long range PCR amplification of the BCR-ABL1 transcript was performed using the Clontech Advantage PCR kit as previously described.^{12 (link)} The cDNA amplicons underwent end-repair and adaptor ligation to generate SMRTbell™ libraries for PacBio sequencing. SMRTbell™ libraries were quantified using the Qubit assay and library size was confirmed using the Agilent DNA 12000 Kit. Each SMRTbell™ amplicon library was loaded on to 1 SMRT cell and sequenced on the PacBio RS II instrument using C4 chemistry and a 120-minute movie time. Circular consensus sequence (CCS) reads were generated for each sample. The CCS reads in FASTQ format were used as input for the automated analysis pipeline.

Schaal W., Ameur A., Olsson-Strömberg U., Hermanson M., Cavelier L, & Spjuth O. (2022). Migrating to Long-Read Sequencing for Clinical Routine BCR-ABL1 TKI Resistance Mutation Screening. Cancer Informatics, 21, 11769351221110872.

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Differential Splicing of Key Genes in Basal-like Tumors

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We have selected 11 genes in total, to be either differentially spliced between basal-like tumours and normal samples (8 genes: AURKA, AURKB, BCL2-a, NEK2, RRM2, TGFBR1, UBE2C, ZBTB16) or differentially spliced in basal-like tumours, between patients with respectively better and worse outcome (4 genes: CCR7, RASSF5, PARP12, TGFBR1). Full length cDNAs were prepared from intact RNA using Primescript Reverse transcriptase (Clontech), oligo dT and a custom transcript switching Oligo (TSO). cDNAs were amplified using semi-nested PCR using the Advantage PCR kit (Clontech) with gene specific primers located near the poly adenylation signal and TSO. Amplified full length cDNAs were analysed using Bioanalyzer DNA7500 kit to size separate all full length isoforms arising from each gene (Additional File 8).

Gracio F., Burford B., Gazinska P., Mera A., Mohd Noor A., Marra P., Gillett C., Grigoriadis A., Pinder S., Tutt A, & de Rinaldis E. (2017). Splicing imbalances in basal-like breast cancer underpin perturbation of cell surface and oncogenic pathways and are associated with patients’ survival. Scientific Reports, 7, 40177.

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TCR Sequencing from mRNA Isolation

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Briefly, the TCR was sequenced by isolating mRNA using RNAeasy Mini Kit (Qiagen, Germany) and synthesized cDNA using SMARTer RACE cDNA Amplification kit (Takara), PCR amplified using PCR Advantage Kit (Takara), and run on a 1% agarose gel for PCR band confirmation. The PCR product was then purified and transformed with TOP10 competent cells (Thermo Fisher, UK) and plated on Luria broth (LB) agar media; and colony PCR was performed to amplify product before isolating plasmid DNA using Spin Miniprep Kit (Qiagen, Germany). The resulting purified plasmid DNA was sent for sequencing at 100 nM of concentration.

Abd Hamid M., Yao X., Waugh C., Rosendo-Machado S., Li C., Rostron T., Frankland J., Peng Y, & Dong T. (2020). Defective Interferon Gamma Production by Tumor-Specific CD8+ T Cells Is Associated With 5′Methylcytosine-Guanine Hypermethylation of Interferon Gamma Promoter. Frontiers in Immunology, 11, 310.

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Sequencing T-Cell Receptor CDR3 Regions

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Briefly, the T-cell receptor was sequenced by isolating mRNA from CTLs using the RNAeasy Mini Kit (Qiagen, Germany) and cDNA was synthesized from 500 ng mRNA using the SMARTer RACE cDNA Amplification kit (Takara), as per manufacturer’s instruction. Briefly, cDNA was PCR-amplified for CDR3 region of both alpha and beta TCR chains using the PCR Advantage Kit (Takara) using the following primers for TRAC: 5’- GGAACTTTCTGGGCTGGGGAAGAAGGTGTCTTCTGG-3’ and for TRBC: 5’- TGCTTCTGATGGCTCAAACACAGCGACCT-3’ and run on a 1% agarose gel for PCR band confirmation (TRAV band at 700bp and TRBV band at 500bp). PCR product was then purified using NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Germany) and transformed into TOP10 competent cells (ThermoFisher, UK) before being plated on LB agar media at 50 μL per plate, as per manufacturer’s instructions. Colony PCR was performed using 200 ng of initial PCR product to amplify the product before isolating the plasmid DNA using the Spin Miniprep kit (Qiagen, Germany). The purified plasmid DNA was then sent for sequencing at a 100 nM concentration, performed by Timothy Rostron and John Frankland of the Sequencing Facility, Weatherall Institute of Molecular Medicine, University of Oxford. Sequencing data of T-cell clones are described in the Results section and Supplementary Table S2.

Hamid M.A., Colin-York H., Alham N.K., Browne M., Cerundolo L., Chen J.L., Yao X., Rosendo-Machado S., Waugh C., Maldonado-Perez D., Bowes E., Verrill C., Cerundolo V., Conlon C.P., Fritzsche M., Peng Y, & Dong T. (2019). Self-maintaining CD103+ cancer-specific T cells are highly energetic with rapid cytotoxic and effector responses. Cancer immunology research, 8(2), 203-216.

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TCR-β Repertoire Analysis of CD8+ T Cells

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One million cells from each epitope-specific polyclonal CD8+ T-cell line were harvested and washed three times with Phosphate Buffered Saline. Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Germany), and cDNA was then synthesized from 300 ng RNA using the SMARTer RACE cDNA amplification kit (Takara Bio, Japan) following the manufacturer's instruction. Subsequently, cDNA was amplified for variable regions of the TCR-β chain using the PCR Advantage kit (Takara Bio), with the primer 5′-TGCTTCTGATGGCTCAAACACAGCGACCT-3′ and run on a 1.2% agarose gel for PCR band confirmation (at 500 bp). PCR products were purified using the Monarch DNA Gel Extraction kit (New England BioLabs, USA) and then transformed into TOP10 competent cells (ThermoFisher). Plasmid DNA was extract using the Spin Miniprep kit (Qiagen) followed by Sanger sequencing.

de Silva T.I., Liu G., Lindsey B.B., Dong D., Moore S.C., Hsu N.S., Shah D., Wellington D., Mentzer A.J., Angyal A., Brown R., Parker M.D., Ying Z., Yao X., Turtle L., Dunachie S., Maini M.K., Ogg G., Knight J.C., Peng Y., Rowland-Jones S.L, & Dong T. (2021). The impact of viral mutations on recognition by SARS-CoV-2 specific T cells. iScience, 24(11), 103353.

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