Agencourt ampure xp reagent

Manufactured by Beckman Coulter

Sourced in United States, Canada

Agencourt AMPure XP reagent is a magnetic bead-based solution used for the purification of DNA and RNA samples. It utilizes a simple and efficient protocol to selectively bind nucleic acids to magnetic beads, allowing for the removal of unwanted contaminants and impurities. The reagent provides a convenient and effective way to purify nucleic acid samples for various downstream applications.

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Lab products found in correlation

68 protocols using agencourt ampure xp reagent

Ion Torrent Whole Genome Sequencing

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Library construction was conducted as per the Ion plus fragment library kit (Invitrogen Cat. No 4471252) for whole genome libraries. Total genomic DNA input was 100 ng which was fragmented using Ion shear^TM enzyme mix II enzyme with an average of 400 bp DNA fragment sizes. The fragmented genomic library was cleaned using Agencourt Ampure XP Reagent (Beckman Coulter). The fragmented DNA was quantified using the Qubit DSDNA HsAssay Kit with the Qubit Fluorometer (Invitrogen). Purified DNA fragments were ligated with adapters specific to cleavage site of endonuclease enzyme, followed by the size selection of genomic library using E-gel 2% in order to get fragment size of 350–400 bp. The library was amplified using 10 cycles of PCR for enrichment of adapter ligated fragments and purified with Agencourt Ampure XP Reagent (Beckman Coulter). Template preparation for sequencing was conducted according to the OneTouch Ion™ Template Kit (Life Technologies). The selected PCR products were again used for emulsion PCR, followed by positive bead recovery. Ion Torrent sequencing was conducted using the Ion PGM^TM 400 Sequencing Kit (Life Technologies) on an Ion Torrent Personal Genome Machine (PGM^TM, Life Technologies) using a 318V²-chip (Ion 318^TM chip, Life Technologies).

Iquebal M.A., Tomar R.S., Parakhia M.V., Singla D., Jaiswal S., Rathod V.M., Padhiyar S.M., Kumar N., Rai A, & Kumar D. (2017). Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence. Scientific Reports, 7, 5299.

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Single-Cell Transcriptome Analysis of Fibroblasts

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Total RNA extracted from fibroblast cells was reverse transcribed according to Ion AmpliSeq^™ Transcriptome Human Gene Expression kit Preparation protocol (Revision A.0; Thermo Fisher Scientific). The cDNA was amplified using Ion AmpliSeq^™ Transcriptome Human Gene Expression core panel (Thermo Fisher Scientific), and the primer sequences were then partially digested, before ligation of Ion P1 Adapters and Ion Xpress^™ Barcode Adapters (Thermo Fisher Scientific). Adaptor‐ligated amplicons were purified using Agencourt^® AMPure^® XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum^® PCR SuperMix High Fidelity and Library Amplification Primer Mix; Thermo Fisher Scientific) and amplified. Size selection and purification were conducted using Agencourt^® AMPure^® XP reagent (Beckman Coulter). The amplicons were quantified using Agilent^® Bioanalyzer^™ instrument with Agilent^® High Sensitivity DNA kit (Agilent).
Samples were then pooled five and five, followed by template preparation on the Ion Chef system using the Ion PI Hi‐Q Chef Kit (Thermo Fisher Scientific). Samples were sequenced on the Ion Proton^™ system using the Ion PI Hi‐Q Sequencing 200 Kit on Ion PI v3 chips (200 bp read length; Thermo Fisher Scientific).

Zhao J.J., Halvardson J., Knaus A., Georgii‐Hemming P., Baeck P., Krawitz P.M., Thuresson A, & Feuk L. (2017). Reduced cell surface levels of GPI‐linked markers in a new case with PIGG loss of function. Human Mutation, 38(10), 1394-1401.

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Ion Amplicon Library Preparation

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Using the Ion Plus Fragment Library Kit, library preparation was performed from 100 ng pooled amplicons for each sample. Briefly, 79 µL of each purified, pooled PCR product was end-repaired at room temperature for 20 min, using 1 µL end-repair enzyme and 20 µL end-repair buffer in a final volume of 100 µL. The end-repaired products were purified with Agencourt™ AMPure™ XP reagent (Beckman Coulter, Brea, CA, USA) and ligated to 2 µL IonCode™ Barcode Adapters (Thermo Fisher Scientific, Loughborough, UK). The adapter-ligated, barcoded libraries were further purified with Agencourt™ AMPure™ XP reagent (Beckman Coulter, Brea, CA, USA) and quantified using the Ion Universal Library Quantitation Kit. qPCR amplification was performed using the StepOnePlus™ real-time PCR system (Thermo Fisher Scientific, Loughborough, UK), and library fragment size distributions were assessed on the LabChip^® GXII Touch (PerkinElmer, Waltham, MA, USA), using the X-mark chip and HT DNA NGS 3K reagent kit according to the manufacturer’s protocol.

Eribo O.A., Naidoo C.C., Theron G., Walzl G., du Plessis N, & Chegou N.N. (2023). An Archetypical Model for Engrafting Bacteroides fragilis into Conventional Mice Following Reproducible Antibiotic Conditioning of the Gut Microbiota. Microorganisms, 11(2), 451.

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Ion AmpliSeq Transcriptome Profiling Protocol

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Libraries were prepared from 50 ng of RNA per sample using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific). Targets of 20,802 genes were amplified with Ion AmpliSeq™ Transcriptome Human Gene Expression core panel (Life Technologies). The primer sequences were then digested, and DNA adaptors (Ion P1 Adaptor and Ion Xpress Barcode Adaptor, Life Technologies) were ligated to the targets. Adaptor ligated targets were purified using the Agencourt AMPure XP reagent (Beckman Coulter) and eluted into an amplification mix containing Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix (Life Technologies) for further amplification. Size-selection purification was performed using Agencourt AMPure XP reagent (Beckman Coulter). Amplicons were quantified using a Fragment Analyzer^TM instrument with a DNF-474 High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies, INC.). Samples were then pooled together with four samples per pool and performed an emulsion PCR on the Ion Chef System using the Ion PI Hi-Q Chef Kit (Life Technologies). The emulsion PCR samples were loaded on Ion PI v3 chips and sequenced on an Ion Proton System using an Ion PI Hi-Q Sequencing 200 Kit chemistry (Life Technologies) to obtain approximately 200 bp read length.

Yimthin T., Cliff J.M., Phunpang R., Ekchariyawat P., Kaewarpai T., Lee J.S., Eckold C., Andrada M., Thiansukhon E., Tanwisaid K., Chuananont S., Morakot C., Sangsa N., Silakun W., Chayangsu S., Buasi N., Day N., Lertmemongkolchai G., Chantratita W., Eoin West T, & Chantratita N. (2021). Blood transcriptomics to characterize key biological pathways and identify biomarkers for predicting mortality in melioidosis. Emerging Microbes & Infections, 10(1), 8-18.

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Next-Gen Sequencing Library Preparation

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Library preparation from 100 ng PCR product per sample was performed using the NEXTflex DNA Sequencing Kit (Bio Scientific Corporation) according to the manufacturer's protocol.
Approximately 40 µl from each purified PCR product was end-repaired at 22 °C for 30 min using 3 µl End-repair enzyme mix and 7 µl End-repair buffer in a final volume of 50 µl. The end-repaired products were purified with 1.8x volume Agencourt™ AMPure™ XP reagent (Beckman Coulter). About 19 µl purified, end-repaired product was ligated to 4 µl IonCode™ Barcode Adapter (ThermoFisher Scientific) with the addition of 31.5 µl Ligation mix at 22 °C for 15 min. The adapted-ligated, barcoded libraries were then purified with 1.8x Agencourt™ AMPure™ XP reagent (Beckman Coulter) and quantified using the Ion TaqMan Library Quantitation Kit (ThermoFisher Scientific). Using the StepOnePlus™ Real-time PCR system (ThermoFisher Scientific), qPCR amplification was performed. Library fragment size distributions were assessed on the LabChip® GXII Touch (PerkinElmer, Waltham, MA, USA), using the X-mark chip and HT DNA NGS 3L reagent kit according to the manufacturer's protocol.

Wagener C., Mohanty N.P., & Measey J. (2020). The gut microbiome facilitates ecological adaptation in an invasive vertebrate.

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Ion Exome Sequencing Library Preparation

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An exome amplicon library was prepared using the Ion AmpliSeq Exome RDY kit (Thermo Fisher, Waltham, MA, USA). Briefly, 100 ng of genomic DNA was added to dehydrated, ultra-high multiplexed primer pairs (12 pools) in a 96-well plate and amplified with the following PCR conditions: 99 °C for 2 min; 99 °C for 15 s and 60 °C for 16 min (10 cycles); and holding at 10 °C. Primers were partially digested using a FuPa reagent, and then sequencing motifs and barcodes were ligated to the amplicons. The library was purified using the Agencourt AMPure XP Reagent (Beckmann Coulter, Brea, CA, USA). The concentration of the final library was determined using the Ion Library TaqMan Quantitation Kit (Thermo Fisher, Waltham, MA, USA) on an ABI 7500 qPCR instrument with the absolute quantification method.
Template preparation was performed with the Ion 540 OT2 Kit (Thermo Fisher, Waltham, MA, USA) on semi-automated Ion OneTouch 2 instrument using the emPCR method. After breaking the emulsion, non-templated beads were removed from the solution during the enrichment process on the Ion OneTouch ES (Thermo Fisher, Waltham, MA, USA) machine. Subsequently, the sequencing primer and polymerase were added, the fully prepared Ion sphere particles were loaded into an Ion 540 chip, and sequencing runs were performed using the Ion S5 Sequencing kit (Thermo Fisher, Waltham, MA, USA) with 500 flows.

Göblös A., Varga E., Farkas K., Árvai K, & Kemény L. (2021). Genetic Investigation of Inverse Psoriasis. Life, 11(7), 654.

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Targeted Cancer Gene Mutation Profiling

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Amplicon libraries were prepared for 10 nm of individual genomic DNA using an Ion AmpliSeq CHPv2 and Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific). Multiplex PCR was carried out for 20 cycles according to the manufacturer’s protocol. The CHPv2 targeted 2790 Catalogue of Somatic Mutations in Cancer mutational hotspot regions in the following 50 cancer‐related genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL.
Sequencing adapters were joined to the amplification products using unique barcodes (Ion Xpress Barcode Adapters 1‐16 Kit; Thermo Fisher Scientific) and purified using Agencourt AMPure XP Reagent (Beckman Coulter) according to the manufacturer’s protocol.²⁴

Yokose T., Kitago M., Matsuda S., Sasaki Y., Masugi Y., Nakamura Y., Shinoda M., Yagi H., Abe Y., Oshima G., Hori S., Yusuke F., Nakano Y., Endo Y., Abe K., Tokino T, & Kitagawa Y. (2020). Combination of KRAS and SMAD4 mutations in formalin‐fixed paraffin‐embedded tissues as a biomarker for pancreatic cancer. Cancer Science, 111(6), 2174-2182.

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Bacterial 16S rRNA Gene Amplification

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PCR was performed for each fecal sample at total reaction volumes of 25 μL containing 2.5 μL of 10X Ex Taq buffer, 2 μL of 2.5 mM dNTP mix, 0.25 μL of Takara Ex Taq DNA Polymerase (5 U/μL), 2 μL of primer pair 515F-806R (10 pM, respectively), and 4 μL of the genomic DNA (gDNA) of the sample. The PCR primers flanked the V4 hypervariable region of the bacterial 16S rRNAs, and their sequences are represented in Table A1. The targeted gene was amplified in a Veriti™ 96-Well Fast Thermal Cycler (Applied biosystems, Woburn, MA, USA). The PCR conditions are as follows: initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final elongation at 72 °C for 5 min. The PCR products were purified with magnetic bead-based Agencourt AMPure XP Reagent (Beckman Coulter Inc., Brea, CA, USA) and DNA quality was evaluated using 1% agarose gel electrophoresis. The final DNA concentration was determined using a Qubit 2.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Mixed amplicons were pooled in equimolar amounts. Single-end sequencing (1 × 300 bp) was carried out with an Illumina iSeq Sequencing system (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions [23 (link),24 ].

Ko Y.S., Tark D., Moon S.H., Kim D.M., Lee T.G., Bae D.Y., Sunwoo S.Y., Oh Y, & Cho H.S. (2023). Alteration of the Gut Microbiota in Pigs Infected with African Swine Fever Virus. Veterinary Sciences, 10(5), 360.

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Metagenomics analysis of bat guano viruses

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Multiple haplotypes of the viruses and their host were expected to be present within the same guano samples. Therefore, the NGS was used to sequence the host and viral PCR products. The PCR products mentioned above were purified with the Agencourt AMPure XP reagent (Beckman Coulter, Brea, Calif., USA). An NGS library was constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs, Massachusetts, USA). Sequencing of paired ends was performed using the MiSeq Reagent Kit v2 (300 cycles) (Illumina®, San Diego, CA, USA), wherein each read was exported from MiSeq Reporter software in FASTQ format, and the NGS data were analyzed using CLC Genomics Workbench software, version 10.1.1 (Filgen, Nagoya, Japan). The overlapped sequence from each end was connected (Mismatch cost = 2, Gap cost = 3, max unaligned mismatches = 0, Minimum score = 6). Then, the sequences were mapped onto reference sequences obtained from the NCBI database. The mapped sequences with 96.44%-100%, similarity to each other were defined as one haplotype of M fuliginosus D-loop DNA [32 (link)]. On the other hand, the viral sequences with 100% similarity to each other were defined as one haplotype of either BtAdV or bat alphaCoV. GenBank accession numbers of all sequencing data of the mitochondrial D-loop DNA, and both of the viruses were presented in Tables 1 and 2, respectively.

Kimprasit T., Nunome M., Iida K., Murakami Y., Wong M.L., Wu C.H., Kobayashi R., Hengjan Y., Takemae H., Yonemitsu K., Kuwata R., Shimoda H., Si L., Sohn J.H., Asakawa S., Ichiyanagi K., Maeda K., Oh H.S., Mizutani T., Kimura J., Iida A, & Hondo E. (2021). Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia. PLoS ONE, 16(1), e0244006.

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Comprehensive Genetic and Protein Analysis

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DNA sequencing was performed in a Clinical Laboratory Improvement Amendments-certified laboratory. Due to limited tissue availability, only 5 samples were retrieved. DNA was extracted using the ARCTURUS PicoPure DNA extraction kit (ThermoFisher Scientific, Grand Island, NY, USA) and purified with Agencourt AMPure XP reagent (Beckman Coulter, Inc., Brea, CA, USA). Targeted sequencing for a panel (46 genes in the first sample and 50 in subsequent samples; Ion AmpliSeq Cancer Hotspot Panel v2, ThermoFisher Scientific) (Supplementary Table 1) was performed on an Ion Torrent platform in 4 of 5 samples [29 (link)]. A data analysis was performed using Torrent Suite (v.4.2.1, ThermoFisher Scientific) and in-house-developed software, OncoSeek. The sequences were aligned to HG19. Mutations were called at an allelic frequency of ≥10 % and confirmed with IGV (v.2.3.1, Broad Institute, Cambridge, MA, USA).
Five samples were retrieved and stained with EGFR and PDGFRA antibodies in a Clinical Laboratory Improvement Amendments-certified laboratory. Five micron-thick formalin-fixed, paraffin-embedded tissues were stained with EGFR (mouse monoclonal, clone 31G7, dilution 1:50; ThermoFisher Scientific) and PDGRA (C-20) (rabbit polyclonal, catalog #sc-338, dilution 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) on a Leica Bond automatic immunostainer (Leica, Buffalo Grove, IL, USA).

Maher O.M., Khatua S., Mukherjee D., Olar A., Lazar A., Luthra R., Liu D., Wu J., Ketonen L, & Zaky W. (2015). Primary intracranial soft tissue sarcomas in children, adolescents, and young adults: single institution experience and review of the literature. Journal of neuro-oncology, 127(1), 155-163.

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