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Agencourt dnadvance genomic dna isolation kit

Manufactured by Beckman Coulter
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Sourced in Australia, United States

The Agencourt DNAdvance Genomic DNA Isolation Kit is a product designed for the efficient extraction and purification of genomic DNA from various biological samples. It utilizes a magnetic bead-based technology to capture and isolate DNA, providing a simple and reliable method for DNA extraction.

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Lab products found in correlation

Qiaxcel capillary electrophoresis instrument, by Qiagen (5 mentions) Str profiling, by American Type Culture Collection (4 mentions) 4d nucleofector, by Lonza (4 mentions) Phusion hot start flex dna polymerase, by New England Biolabs (4 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (3 mentions) Qubit 3.0 fluorometric quantitation, by Thermo Fisher Scientific (3 mentions) Purelink genomic dna isolation, by Thermo Fisher Scientific (3 mentions) Truseq custom amplicon v1.5 targeted resequencing workflow, by Illumina (3 mentions) Miseq instrument, by Illumina (3 mentions) Miseq system, by Illumina (3 mentions) Poly d lysine coated plate, by Corning (2 mentions) Se cell line kit, by Lonza (2 mentions) Agencourt ampure xp cleanup, by Beckman Coulter (2 mentions) Phusion hot start 2 dna polymerase, by Thermo Fisher Scientific (2 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Se cell line nucleofector kit, by Lonza (2 mentions) Advanced dmem, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Agencourt DNAdvance kit (26 citations) DNAdvance kit (16 citations) DNAdvance Genomic DNA Isolation kit (8 citations) Agencourt DNAdvance (6 citations) DNA Agencourt DNAdvance Kit (2 citations) DNAdvance genomic DNA kit (2 citations)

31 protocols using agencourt dnadvance genomic dna isolation kit

1

CRISPR/base editor gene editing

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HEK293T cells grown in the absence of antibiotic were seeded on 48-well poly-D-lysine coated plates (Corning). After 12–14 h, cells were transfected at ~70% confluency with 750 ng of Cas9 or base editor plasmid, 250 ng of sgRNA expression plasmid, 1.5 μL of Lipofectamine 3000 (Thermo Fisher Scientific), and for HDR assays 0.7 μg of single-stranded donor DNA template (100 nt, PAGE-purified from IDT) according to the manufacturer’s instructions. 100-mer single-stranded oligonucleotide donor templates are listed in Supplementary Table 10.
Genomic DNA was harvested 48 h post-transfection (as described by Tessier-Lavigne et. al. during the development of the CORRECT method42 (link)) using the Agencourt DNAdvance Genomic DNA isolation Kit (Beckman Coulter) according to the manufacturer’s instructions. A size-selective DNA isolation step ensured that there was no risk of contamination by the single-stranded donor DNA template in subsequent PCR amplification and sequencing steps. We re-designed amplification primers to ensure there was minimal risk of amplifying donor oligo template.
Gaudelli N.M., Komor A.C., Rees H.A., Packer M.S., Badran A.H., Bryson D.I, & Liu D.R. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551(7681), 464-471.
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2

CRISPR Editing of U2OS Cells

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U2OS cells (a gift from Toni Cathomen, Freiburg) and U2OS.EGFP cells (containing a single integrated copy of an EGFP-PEST reporter gene)30 (link) were cultured in Advanced DMEM supplemented with 10% HI FBS, 2 mM GlutaMax, and penicillin/streptomycin at 37°C with 5% CO2. The growth media for U2OS.EGFP cells was additionally supplemented with 400 µg ml−1 Geneticin. All cell culture reagents were obtained from Life Technologies. Cell line identity was validated by STR profiling (ATCC) and deep-sequencing, and cells were tested bi-weekly for mycoplasma contamination. Unless otherwise noted, cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid. For negative control experiments, Cas9 plasmids were co-transfected with a U6-null plasmid. Nucleofections were performed using the DN-100 program on a Lonza 4-D Nucleofector with the SE Cell Line Kit according to the manufacturer’s protocol (Lonza). For T7E1 assays, GUIDE-seq experiments, and targeted deep sequencing, genomic DNA was extracted ~72 hours post-transfection using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics).
Kleinstiver B.P., Pattanayak V., Prew M.S., Tsai S.Q., Nguyen N., Zheng Z, & Joung J.K. (2016). High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets. Nature, 529(7587), 490-495.
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3

DNA Extraction and Telomere Length Measurement

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DNA was extracted from ear tissue using the Agencourt DNAdvance Genomic DNA isolation kit (Beckman Coulter, Mount Waverly, Australia). All samples were quantified using a Qubit 2.0 fluorometer (ThermoFisher, Waltham, MA) and Qubit dsDNA High Sensitivity Assay Kit prior to carrying out telomere length measurements using quantitative PCR.
Smith L.E., Jones M.E., Hamede R., Risques R., Patton A.H., Carter P.A, & Storfer A. (2020). Telomere length is a susceptibility marker for Tasmanian devil facial tumor disease. EcoHealth, 17(3), 280-291.
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4

Hexaploid Wheat Genome Diversity Analysis

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As an initial methodology study, the unpublished wheat allohexaploid WEC sequence data generated by the Canadian Triticum Applied Genomics (CTAG2) was used. Briefly, 114 diverse hexaploid wheat lines representing the genetic diversity of a large wheat population in Canada were collected. Genomic DNA was extracted from leaf tissue for each accession using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) and subjected to sequence capture using the NimbleGen SeqCap EZ wheat whole-genome assay to generate a sequence capture library [3 (link)]. The sequence capture library was sequenced on an Illumina HiSeq2000 instrument to generate about 30 million reads per accession. Three data sets (raw read fastq file, preprocessed with quality trimming, and duplicate removal) generated from one sample were used for variant calling tool evaluation before a whole population diversity was investigated. The experiment design and workflow were depicted in Fig. 1.
Yao Z., You F.M., N’Diaye A., Knox R.E., McCartney C., Hiebert C.W., Pozniak C, & Xu W. (2020). Evaluation of variant calling tools for large plant genome re-sequencing. BMC Bioinformatics, 21, 360.
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5

CRISPR Editing of U2OS Cells

Cited 1 time
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U2OS cells (a gift from Toni Cathomen, Freiburg) and U2OS.EGFP cells (containing a single integrated copy of an EGFP-PEST reporter gene)30 (link) were cultured in Advanced DMEM supplemented with 10% HI FBS, 2 mM GlutaMax, and penicillin/streptomycin at 37°C with 5% CO2. The growth media for U2OS.EGFP cells was additionally supplemented with 400 µg ml−1 Geneticin. All cell culture reagents were obtained from Life Technologies. Cell line identity was validated by STR profiling (ATCC) and deep-sequencing, and cells were tested bi-weekly for mycoplasma contamination. Unless otherwise noted, cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid. For negative control experiments, Cas9 plasmids were co-transfected with a U6-null plasmid. Nucleofections were performed using the DN-100 program on a Lonza 4-D Nucleofector with the SE Cell Line Kit according to the manufacturer’s protocol (Lonza). For T7E1 assays, GUIDE-seq experiments, and targeted deep sequencing, genomic DNA was extracted ~72 hours post-transfection using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics).
Kleinstiver B.P., Pattanayak V., Prew M.S., Tsai S.Q., Nguyen N., Zheng Z, & Joung J.K. (2016). High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets. Nature, 529(7587), 490-495.
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6

Measuring CRISPR-Cas9 Mutagenesis Frequencies

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Roughly 72 hr post-transfection, genomic DNA was extracted from U2OS cells using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics), and T7 endonuclease I (T7E1) assays were performed as previously described26 (link). Briefly, 600–800 nt amplicons surrounding on-target sites were amplified from ~100 ng of genomic DNA using Phusion Hot-Start Flex DNA Polymerase (New England Biolabs, NEB) using the primers listed in Supplementary Table 3. PCR products were visualized (using a QIAxcel capillary electrophoresis instrument, Qiagen), and purified (Agencourt Ampure XP cleanup, Beckman Coulter Genomics), Denaturation and annealing of ~200 ng of the PCR product was followed by digestion with T7EI (NEB). Digestion products were purified (Ampure) and quantified (QIAxcel) to approximate the mutagenesis frequencies induced by Cas9-sgRNA complexes.
Chen J.S., Dagdas Y.S., Kleinstiver B.P., Welch M.M., Sousa A.A., Harrington L.B., Sternberg S.H., Joung J.K., Yildiz A, & Doudna J.A. (2017). Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature, 550(7676), 407-410.
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7

Targeted Sequencing from Transfected Cells

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Transfected cells were harvested after 3 d. The genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer’s instructions. Genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Information. PCR amplification was carried out with Phusion hot-start II DNA polymerase (ThermoFisher) according to the manufacturer’s instructions. PCR products were purified using RapidTips (Diffinity Genomics). Secondary PCR was performed to attach sequencing adaptors. The products were gel-purified and quantified using the KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described1 (link).
Kim Y.B., Komor A.C., Levy J.M., Packer M.S., Zhao K.T, & Liu D.R. (2017). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nature biotechnology, 35(4), 371-376.
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8

Metagenomics of Particle-Attached Communities

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DNA extractions were performed for twelve 175-µL-volume aliquots of the initial, unincubated seawater and for particles harvested after 34.5, 59, 103, 116.75, 113, 153.5, and 166.5 h of incubation (see Dataset S5 for sample metadata; in total, metagenomes from 495 particle-attached communities were analyzed in this study). DNA was extracted from all samples with the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter; modifications noted in SI Appendix, Extended Methods). Metagenomic libraries were prepared with the Nextera XT DNA Library Prep Kit and index primers (Illumina) using the protocol developed by Rinke et al. (18 (link)) for low DNA inputs (SI Appendix, Extended Methods). Libraries were quantified on an Agilent 4200 TapeStation system with High Sensitivity D5000 ScreenTapes (Agilent Technologies) and pooled by time point in equimolar amounts. Sequencing was performed on an Illumina HiSeq 2500 machine (250-bp paired-end reads) at the Whitehead Institute for Biomedical Research.
Szabo R.E., Pontrelli S., Grilli J., Schwartzman J.A., Pollak S., Sauer U, & Cordero O.X. (2022). Historical contingencies and phage induction diversify bacterioplankton communities at the microscale. Proceedings of the National Academy of Sciences of the United States of America, 119(30), e2117748119.
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9

Targeted Genomic DNA Sequencing

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Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. On-target and off-target genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion high-fidelity DNA polymerase (ThermoFisher) according to the manufacturer's instructions using 5 ng of genomic DNA as a template. Cycle numbers were determined separately for each primer pair as to ensure the reaction was stopped in the linear range of amplification (30, 28, 28, 28, 32, and 32 cycles for EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 primers, respectively). PCR products were purified using RapidTips (Diffinity Genomics). Purified DNA was amplified by PCR with primers containing sequencing adaptors. The products were gel-purified and quantified using the Quant-iT™ PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described.32
Komor A.C., Kim Y.B., Packer M.S., Zuris J.A, & Liu D.R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420-424.
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10

CRISPR Plasmid Transfection in U2OS and HEK293 Cells

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U2OS cells (obtained from Toni Cathomen, Freiburg) and HEK293 cells were cultured at 37°C with 5% CO2 in Advanced DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax, and penicillin/streptomycin (all cell culture products were obtained from Life Technologies). Cell line identity was validated by STR profiling (ATCC) and mycoplasma testing was performed twice per month. 2 × 105 cells were transfected using the SE Cell Line Nucleofector Kit with 500 ng Cpf1- or SpCas9-encoding plasmid and 250 ng gRNA expression plasmid and the DN-100 program for U2OS cells, or with 300 ng Cpf1- or SpCas9-encoding plasmid and 150 ng gRNA expression plasmid using the CM-137 program for HEK293 cells, on a 4D-Nucleofector according to the manufacturer’s instructions (Lonza). For negative control transfections, Cpf1- or SpCas9-encoding plasmids were co-transfected with a U6-null plasmid that did not encode a crRNA. Genomic DNA extraction was performed approximately 72 hours following nucleofection using an Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter).
Kleinstiver B.P., Tsai S.Q., Prew M.S., Nguyen N.T., Welch M.M., Lopez J.M., McCaw Z.R., Aryee M.J, & Joung J.K. (2016). Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature biotechnology, 34(8), 869-874.
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