Ampure xp beads

Manufactured by Agilent Technologies

Sourced in United States

AMPure XP beads are a magnetic bead-based DNA purification product. They are designed to selectively bind DNA fragments based on their size, enabling the purification and recovery of DNA samples from various sample types.

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AMPure beads (13 citations) AMPure XP (9 citations) AMPure XP magnetic beads (3 citations)

61 protocols using ampure xp beads

Kapa Hyper Prep Kit Protocol for Targeted Sequencing

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1 µg of genomic DNA per sample was processed using the Kapa Hyper Prep Kit following manufacturer’s recommended protocol (Kapa Biosystems, Inc., Wilmington, MA, USA). Briefly, samples were enzymatically sheared, A-tailed, and ligated to standard Illumina dual indexed adapters. Libraries were purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) and amplified in a Bio-Rad T100 Thermocycler (Bio-Rad, Hercules, CA, USA). Libraries were again purified using AMPure XP beads and validated with the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA). Libraries were then hybridized to biotinylated target probes for the DBA following manufacturer’s recommended protocol (IDT). After hybridization, the captured DNA was captured with magnetic streptavidin beads and purified with several wash buffers in order of decreasing stringency. Libraries were again briefly amplified and purified using AMPure XP beads before final validation on with the TapeStation D1000 ScreenTape (Agilent Technologies). Up to 96 samples were normalized and pooled together for sequencing. Sequencing was conducted on the Illumina NextSeq 500 using 150-bp paired-end reads (Illumina Inc, San Diego, CA, USA).

Richardson M.E., Chong H., Mu W., Conner B.R., Hsuan V., Willett S., Lam S., Tsai P., Pesaran T., Chamberlin A.C., Park M.S., Gray P., Karam R, & Elliott A. (2018). DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes. Genetics in Medicine, 21(3), 683-693.

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Bacterial 16S rRNA Sequencing from Caecal Samples

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Extraction of the bacterial DNA from the caecal digesta samples and high-throughput sequencing of the V3–V4 hypervariable region of the bacterial 16S rRNA gene was performed on an Illumina MiSeq platform according to their standard protocols (Eurofins Genomics, Ebersberg, Germany). Briefly, the V3–V4 region was PCR-amplified using universal primers containing adapter overhang nucleotide sequences for forward and reverse index primers. Amplicons were purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN) and set up for the index PCR with Nextera XT index primers (Illumina, San Diego, CA). The indexed samples were purified using AMPure XP beads, quantified using a fragment analyser (Agilent, Santa Clara, CA), and equal quantities from each sample were pooled. The resulting pooled library was quantified using the Bioanalyzer 7500 DNA kit (Agilent, Santa Clara, CA) and sequenced using the v3 chemistry (2 × 300 bp paired-end reads).

Dowley A., O’Doherty J.V., Mukhopadhya A., Conway E., Vigors S., Maher S., Ryan M.T, & Sweeney T. (2022). Maternal supplementation with a casein hydrolysate and yeast beta-glucan from late gestation through lactation improves gastrointestinal health of piglets at weaning. Scientific Reports, 12, 17407.

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Extraction and Sequencing of Bacterial 16S rRNA

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Genomic DNA was extracted using an E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, United States) according to the manufacturer’s protocols. The concentrations and purity of the resultant DNA were determined using a NanoDrop ND-2000 (NanoDrop, United States), and the quality was assessed by running aliquots on gels. The samples were stored at –80°C for further analysis.
The 16S rRNA gene was amplified by polymerase chain reaction (PCR) with the primers 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), which target the hypervariable V3–V4 region of the bacterial 16S rRNA gene. PCR was performed in triplicate with Phusion High-Fidelity PCR Master Mix (New England Biolabs) using 30 ng of template DNA. The PCR products were purified with AMPure XP beads and quantified/qualified with an Agilent 2100 Bioanalyzer (Agilent, CA, United States). PCR products from different samples were mixed equally and used to construct an Illumina paired-end library with a NEBNext Ultra™ DNA Library Prep Kit for Illumina (NE, United States). Then, the amplicon library was sequenced in paired-end mode (2 × 300 bp) on an Illumina MiSeq platform (Illumina, San Diego, CA, United States) according to standard protocols.

Yang Y., Zhao M., He X., Wu Q., Li D.L, & Zang W.J. (2021). Pyridostigmine Protects Against Diabetic Cardiomyopathy by Regulating Vagal Activity, Gut Microbiota, and Branched-Chain Amino Acid Catabolism in Diabetic Mice. Frontiers in Pharmacology, 12, 647481.

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Exome Capture Library Preparation

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DNA library construction was performed according to the Agilent SureSelect^XT Target Enrichment System for Illumina Pair-End Sequencing Library protocol with modifications. Briefly, 500ng input DNA was used for each sample and sheared as described above. Samples were then purified using Agencourt AMPure XP beads and quality assessed using an Agilent 2100 Bioanalyzer system. The ends were repaired, the product bead-purified and A bases added to the 3′ ends of the DNA fragments to generate an A-base overhang. The product of this reaction was again bead purified, and to this indexing-specific paired-end adaptors were attached using T4 ligase. The product of this reaction was bead-purified and the adaptor-ligated library amplified using Herculase II fusion DNA polymerase. The post-amplification product was bead purified and assesses for quality using the Agilent Bioanalyzer system. The library was then hybridized with the SureSelect^XT Human All Exon 50Mb library to perform exome capture. Hybridization was performed 65°C for 16 hours and the exome capture library enriched using magnetic Dynabeads (Agilent). Index tags were added by post-hybridization amplification. Exome capture libraries were sequenced on an Illumina HiSeq 2000 using the standard Illumina SOP (21 ) with modifications.

Tricoli J.V., Boardman L.A., Patidar R., Sindiri S., Jang J.S., Walsh W.D., McGregor PM I.I.I., Camalier C.E., Mehaffey M.G., Furman W.L., Bahrami A., Williams P.M., Lih C.J., Conley B.A, & Khan J. (2017). A Mutational Comparison of Adult and Adolescent and Young Adult (AYA) Colon Cancer. Cancer, 124(5), 1070-1082.

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Microbial DNA Extraction and 16S rRNA Sequencing

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Bacterial total DNA was extracted from the collected fecal sample using the HiPure Stool DNA Kit (Magen, Guangzhou, China) according to the manufacturer's instructions. The concentration and purity of the extracted DNA were determined by NanoDrop (Thermo Fisher, UD, USA) and agarose gel electrophoresis, respectively. V3–V4 region of 16s rDNA was selected and PCR amplified with the primer pairs: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT. The PCR cycling conditions was as follows, first round: initial denaturation at 94 °C for 2 minutes, 35 cycles of 98 °C for 10 seconds, 62 °C for 30 seconds and 68 °C for 30 seconds, followed by a final extension at 68 °C for 5 minutes. The PCR amplicons were purified with AmpureXp beads (Beckman Coulter, Life Sciences, CA, USA) and applied for the second round PCR amplification. Second round: initial denaturation at 94 °C for 2 minutes, 15 cycles of 98 °C for 10 seconds, 65 °C for 30 seconds and 68 °C for 30 seconds, followed by a final extension at 68 °C for 5 minutes. The amplicons of the second round PCR were purified with AmpureXp beads and quantified with Bioanalyzer DNA 1000 chip (Agilent Technologies, CA, USA). The DNA libraries were pair-end sequenced with the sequencing strategy PE300 by the MiSeq system (Illumina, San Diego, CA, USA).

Dong L., Han L., Duan T., Lin S., Li J, & Liu X. (2020). Integrated microbiome–metabolome analysis reveals novel associations between fecal microbiota and hyperglycemia-related changes of plasma metabolome in gestational diabetes mellitus. RSC Advances, 10(4), 2027-2036.

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Bacterial 16S rRNA Gene Sequencing

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High-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed on an Illumina MiSeq platform according to their standard protocols (Eurofins, Wolverhampton, UK). Briefly, the V3-V4 region was PCR-amplified using universal primers containing adapter overhang nucleotide sequences for forward and reverse index primers. Amplicons were purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) and set up for the index PCR with Nextera XT index primers (Illumina, San Diego, CA, United States). The indexed samples were purified using AMPure XP beads, quantified using a fragment analyzer (Agilent, Santa Clara, CA, USA), and equal quantities from each sample were pooled. The resulting pooled library was quantified using the Bioanalyzer 7500 DNA kit (Agilent) and sequenced using the v3-v4 chemistry (2 × 300 bp paired-end reads).

Rattigan R., Sweeney T., Vigors S., Thornton K., Rajauria G, & O’Doherty J.V. (2019). The Effect of Increasing Inclusion Levels of a Fucoidan-Rich Extract Derived from Ascophyllum nodosum on Growth Performance and Aspects of Intestinal Health of Pigs Post-Weaning. Marine Drugs, 17(12), 680.

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DNA Extraction and Library Prep

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Extracted DNA was purified using Ampure XP beads (Agilent). Fifty microliters of DNA was vortexed with 90μl Ampure XP beads and washed twice with 80% ethanol before elution in 25μl molecular water. DNA was normalized to 0.2ng/μl and libraries prepared from 1μg DNA using the Nextera XT Library preparation kit (Illumina) according to the manufacturer’s instructions. Normalization and QC of libraries was performed using a Qubit 2.0 fluorometer (Thermo Fisher) and 2200 Tapestation (Agilent). Paired-end 300bp sequencing was performed on an Illumina MiSeq, with 10–16 samples multiplexed per sequencing run.

Peto L., Fawcett N.J., Crook D.W., Peto T.E., Llewelyn M.J, & Walker A.S. (2019). Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae. PLoS ONE, 14(11), e0222831.

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Illumina TruSeq ChIP Library Preparation

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Preparation of paired-end sequencing libraries was performed using the Illumina TruSeq ChIP library preparation kit–Set-A (15034288), according to the manufacturer’s guidelines. Ligation products were size-selected (250–300 bp) and purified from a 2% low-melting agarose gel using the MinElute Gel Extraction Kit (Qiagen). Ampure XP beads (Agilent) were used for cleanup steps and size selection. The final purified product was quantitated using Picogreen in a QuantiFluor dsDNA System (Promega). Paired-end sequencing (2 X 150bp) was performed on the Illumina HiSeq 3000 platform at the Max Planck Genome Centre (Cologne, Germany). The ChIP-seq raw data employed in this study are deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), under accession no. GSE138429.

Cardoso da Silva R., Villar-Fernández M.A, & Vader G. (2020). Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase. PLoS Genetics, 16(6), e1008905.

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Bacterial 16S rRNA Sequencing from Caecal Samples

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Extraction of the bacterial DNA from the caecal digesta samples and high-throughput sequencing of the V3–V4 hypervariable region of the bacterial 16S rRNA gene on an Illumina MiSeq platform were performed according to their standard protocols (Eurofins Genomics, Ebersberg, Germany). Briefly, the V3–V4 region was PCR-amplified using universal primers containing adapter overhang nucleotide sequences for forward and reverse index primers. Amplicons were purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN) and set up for the index PCR with Nextera XT index primers (Illumina, San Diego, CA). The indexed samples were purified using AMPure XP beads, quantified using a fragment analyzer (Agilent, Santa Clara, CA), and equal quantities from each sample were pooled. The resulting pooled library was quantified using the Bioanalyzer 7500 DNA kit (Agilent, Santa Clara, CA) and sequenced using the v3 chemistry (2 × 300 bp paired-end reads).

Venardou B., O'Doherty J.V., Vigors S., O'Shea C.J., Burton E.J., Ryan M.T, & Sweeney T. (2021). Effects of dietary supplementation with a laminarin-rich extract on the growth performance and gastrointestinal health in broilers. Poultry Science, 100(7), 101179.

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Targeted Amplicon Sequencing of Solid Tumors

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Library preparation: Libraries were prepared using an AmpliSeq Illumina Focus Panel. It is an amplicon-based targeted panel with DNA and RNA pool-investigating mutations across 52 genes with known relevance to solid tumors. Libraries were prepared as per the protocol described in AmpliSeq for the Illumina Focus Panel Reference Guide. The recommended input for DNA and RNA for the library was 1–100 ng. Because of the disparity in nucleic acid concentration from each sample, based on optimization, samples were normalized with nuclease-free water to attain the final concentration of 90 ng DNA. Low-quantity DNA was taken neat.
PCR amplification of DNA and cDNA was performed. Amplicons were then partially digested and ligated to uniquely brocaded index-adaptor sequences provided by Illumina. Libraries purified with Agencort AMPure XP beads were further analyzed for quality on an Agilent 4200 TapeStation using high-sensitivity D1000 screen tape and a high-sensitivity D1000 reagent
During the library preparation, two more QC check of DNA libraries was performed by using a TapeStation to measure the presence of desired amplicons.

Chougule A., Jagtap V., Nikam A., Kale S., Nambiar K., Bagayatkar P., Chandrani P., Kaushal R., Noronha V., Patil V., Banavali S, & Prabhash K. (2022). Comprehensive Development and Implementation of Good Laboratory Practice for NGS Based Targeted Panel on Solid Tumor FFPE Tissues in Diagnostics. Diagnostics, 12(5), 1291.

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