Dna 12000 kit

Manufactured by Agilent Technologies

Sourced in United States

The DNA 12000 Kit is a lab equipment product from Agilent Technologies designed for the analysis and quantification of DNA samples. It provides a reliable and accurate method for determining the size and concentration of DNA fragments ranging from 100 to 12,000 base pairs.

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Lab products found in correlation

Agilent 2000 bioanalyzer, by Agilent Technologies (2 mentions) Onestep rt pcr kit, by Qiagen (2 mentions) Rnaeasy mini kit, by Qiagen (2 mentions) Bluepippin, by Sage Science (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Pcr advantage kit, by Takara Bio (1 mentions) Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions) Bsai hf, by New England Biolabs (1 mentions) Agencourt ampure xp system, by Beckman Coulter (1 mentions) Quant it dsdna assay kit, by Thermo Fisher Scientific (1 mentions) Plasmid midi kit, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Ecos competent escherichia coli jm109, by Nippon Gene (1 mentions) Thermocycler, by Thermo Fisher Scientific (1 mentions) Ampure pb bead, by Pacific Biosciences (1 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions) Mobio powersoil dna isolation kit, by Qiagen (1 mentions) Hiseq x ten instrument, by Illumina (1 mentions) Nextflex dna sequencing kit, by Bio Scientific (1 mentions) Biomek fxp, by Bio Scientific (1 mentions)

9 protocols using dna 12000 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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Plasmid Cloning and Spike-in Preparation

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Plasmid cloning vectors with spike-in sequence inserts were transformed into ECOS Competent Escherichia coli JM109 (Nippon Gene, Toiya, Japan) following the manufacturer's instructions. Plasmid DNA was extracted from overnight liquid cultures using the QIAGEN Plasmid Midi Kit. Plasmid DNA was then linearized using the following single-cutting restriction enzymes, according to the manufacturer's instructions: BpmI (New England Biolabs) for spike-ins Ec5001, Ec5002, Ec5005, Ec5502 and Ga5501; BsaI-HF (New England Biolabs) for Ec5003, Ec5004, Ec6001, Bv5501, Ca5501 and Tb5501; and ScaI (TaKaRa Bio) for Ec5501. Linearized plasmid DNA was purified using the Agencourt AMPure XP system (Beckman Coulter) and size and integrity were verified by electrophoresis using the Bioanalyzer 2100 with a DNA 12000 Kit (Agilent). DNA concentrations were determined with a high-sensitivity Quant-iT dsDNA Assay Kit (Invitrogen) using a Qubit Fluorometer 3.0 (Life Technologies). Plasmid DNA was diluted to 10 ng/μl in Tris-EDTA (TE) buffer (pH 8.0) and distributed in single-use aliquots stored at −80°C. Spike-in sequences were verified by Sanger sequencing (see Supplementary Data for details) and experimentally determined sequences were in all cases in agreement with designed sequences. Spike-in standard mixes were prepared based on estimated copy numbers and stored in TE buffer at −20°C until use.

Tourlousse D.M., Yoshiike S., Ohashi A., Matsukura S., Noda N, & Sekiguchi Y. (2016). Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing. Nucleic Acids Research, 45(4), e23.

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Competitive RT-PCR Analysis of MCL-1 Expression

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Total RNA was extracted from cells using the RNA Easy mini kit (Qiagen, Cat # 74104), and RNA integrity was evaluated using an Agilent Bioanalyzer 2000. Competitive RT-PCR was performed with a one-step RT-PCR kit (Qiagen, Cat # 210210) using the following primer sets containing an equimolar ratio of MCL-1 (forward, 5′-GGGCAGGATTGTGACTCTCATT- 3′; reverse, 5′-GATGCAGCTTTCTTGGTTTATGG-3′) against β-actin (forward, 5′-TCACCCACACTGTGCCCATCTACGA- 3′; reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′). Reverse transcription was coupled with PCR (25 cycles) on a thermocycler (Eppendorf, Mastercycler® GX2). PCR products were quantified on the Agilent Bioanalyzer 2000 using the DNA 12,000 kit (Cat # 5067-1508). In brief, samples were loaded onto DNA microchips, and the DNA fragments were then separated by capillary electrophoresis. The target DNA sizes and relative quantities were calculated on the basis of DNA ladders and an internal marker, respectively. The associated software then generates agarose gel-like images.

Kawakami H., Huang S., Pal K., Dutta S.K., Mukhopadhyay D, & Sinicrope F.A. (2016). Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer. Molecular cancer therapeutics, 15(12), 3015-3027.

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Competitive RT-PCR for Bcl-xL Expression

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Total RNA was extracted from cells using RNA easy mini kit (Qiagen, Germantown, MD) and RNA integrity was confirmed using an Agilent Bioanalyzer 2000 (Santa Clara, CA). Competitive RT-PCR was performed using a one-step RT-PCR kit (Qiagen) with mixing of Bcl-xL (forward: 5′-GATCCCCATGGCAGCAGTAAAGCAAG-3′, reverse: 5′-CCCCATCCCGGAAGAGTTCATTCACT-3′) and β-actin (forward: 5′-TCACCCACACTGTGCCCATCTACGA-3′, reverse: 5′-CAGCGGAACCGCTCATTGCCAATGG-3′) primers at molar ratio of 1:1. Reverse transcription was coupled with PCR (x 25 cycles) on a thermocycler (Applied Biosystems, Grand Island, NY). PCR products were quantified on the Agilent Bioanalyzer 2000 using the DNA 12,000 kit.

Okamoto K., Zaanan A., Kawakami H., Huang S, & Sinicrope F.A. (2014). Reversal of Mutant KRAS-mediated Apoptosis Resistance by Concurrent Noxa/Bik Induction and Bcl-2/Bcl-xL Antagonism in Colon Cancer. Molecular cancer research : MCR, 13(4), 659-669.

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PacBio Sequencing of Amplified DNA

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The RCA product of the simple community was purified and concentrated with 1× volume AMPure® PB Beads (100-265-900, PacBio, Menlo Park, CA, USA) giving 7 μg of purified product, determined by Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). The purified product was gently sheared (to reduce hyperbranched DNA structures) using Covaris® g-TUBE™ device (Covaris, Woburn, MA, USA) at 6,000 rpm for 1.5 min. To verify the size of fragments after shearing, sheared and unsheared products were run on a 0.5 % (w/v) agarose gel. The sheared RCA product was purified with 0.45× AMPure® Beads and was used to prepare the 10 kb SMRTbell™ template in accordance with the Pacific Biosciences procedure for ‘10 kb Template Preparation and Sequencing’. BluePippin™ (Sage Science, Beverly, MA, USA) was used to select libraries of sizes ranging from 6-10 kb. The distribution of library sizes was first assessed on Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) with the DNA 12000 kit (5067-1509, Agilent Technologies, Inc.) before they were sequenced on a PacBio sequencer, using P5-C3 chemistry coupled with MagBead Standard Seq v2 collection protocol.

Li C., Chng K.R., Boey E.J., Ng A.H., Wilm A, & Nagarajan N. (2016). INC-Seq: accurate single molecule reads using nanopore sequencing. GigaScience, 5, 34.

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Metagenomic DNA Sequencing from Sediment

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DNA was extracted from 10 g of sediment sample for each experiment replicate, using the MoBio PowerSoil DNA Isolation kit (MO BIO Laboratories, USA) according to the manufacturer’s protocol. DNA concentrations of the extract were measured with a Qubit fluorometer. For each library construction, about 100 ng DNA was fragmented with Covaris S2 (Covaris, USA) and was used to construct metagenomic DNA library with NEXTflex™ DNA Sequencing Kit compatible with the Biomek® FXp (Bio Scientific, USA). Notably, PCR amplification was limited to 12 cycles for each Illumina library. The quality of DNA library was examined by Agilent Bioanalyzer 2100 (Agilent, USA) with DNA 12000 Kit. Paired-end Illumina sequencing (2 × 150 bp) was performed for each metagenomic library on Hiseq Xten instruments (Illumina).

Chen P., Zhou H., Huang Y., Xie Z., Zhang M., Wei Y., Li J., Ma Y., Luo M., Ding W., Cao J., Jiang T., Nan P., Fang J, & Li X. (2021). Revealing the full biosphere structure and versatile metabolic functions in the deepest ocean sediment of the Challenger Deep. Genome Biology, 22, 207.

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PacBio Sequencing of Schistocerca gregaria

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The library preparation for PacBio sequencing was performed with a "SMRTbell Template Prep Kit 1.0" according to the PacBio protocol (version 100-286-000). For each of the two libraries, 10 µg of the
S. gregaria high-molecular-weight DNA was used as input in two parallel 50-µl reactions.
For library size selection, a "0.75% Dye-Free Agarose Gel Cassette” (ref: BLF7510) was used on a Blue Pippin (Sage Science) with the "0.75% DF Marker S1 high-pass 15–20kb" protocol for a lower cut-off of 12 kb. Fragment size distribution was determined with a “DNA 12000 kit” (ref: 5067-1508) for the first library and a “Fragment Analyzer (Agilent) - High Sensitivity Large Fragment 50 kb kit” (ref: DNF-464-0500) for the second library. The resulting libraries had an average length of 16.5 and 22 kb, respectively.
No extension time was used for the sequencing as recommended for size selected libraries in the “Quick Reference Card 101-461-600 version 07”. The first run was performed on a PacBio RSII System (V4.0 chemistry, polymerase P6). Fifteen additional runs were performed on a PacBio Sequel system with 2.0 Chemistry, polymerase and SMRTCells. The same conditions were used to sequence 20 more SMRTCells with the second library on the PacBio Sequel system.

Verlinden H., Sterck L., Li J., Li Z., Yssel A., Gansemans Y., Verdonck R., Holtof M., Song H., Behmer S.T., Sword G.A., Matheson T., Ott S.R., Deforce D., Van Nieuwerburgh F., Van de Peer Y, & Vanden Broeck J. (2021). First draft genome assembly of the desert locust, Schistocerca gregaria. F1000Research, 9, 775.

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Bioanalyzer-based DNA and RNA Profiling

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DNA and RNA samples were loaded into the corresponding kits; Agilent DNA 12000 Kit and Agilent RNA 6000 Nano Kit, both designed for use with the Agilent 2,100 Bioanalyzer instrument, according to guide instructions. Results were analyzed with the 2,100 Expert Software.

Martínez J., Lampaya V., Larraga A., Magallón H, & Casabona D. (2023). Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction. Frontiers in Molecular Biosciences, 10, 1248511.

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Bulk RNA-seq Analysis Pipeline

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A total of 1 µg of RNA per sample was used to synthesize 50-bp-long single-end mRNA libraries with an Illumina TruSeq Stranded mRNA Library Prep Kit. The integrity and quantity of the libraries were determined on the Bioanalyzer using the DNA 12000 Kit (Agilent). The barcoded libraries were pooled and sequenced on an Illumina HiSeq 4000, with an average depth of 20 million reads per sample. The raw reads were mapped to the human genome build GRCh38, and gene level counts were determined using Spliced Transcripts Alignment to a Reference, version 2.5 (71 (link)). Subsequent data processing followed the method outlined in ref. 72 (link).

Madsen R.R., Knox R.G., Pearce W., Lopez S., Mahler-Araujo B., McGranahan N., Vanhaesebroeck B, & Semple R.K. (2019). Oncogenic PIK3CA promotes cellular stemness in an allele dose-dependent manner. Proceedings of the National Academy of Sciences of the United States of America, 116(17), 8380-8389.

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