Dynabeads kilobasebinder kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Dynabeads kilobaseBINDER kit is a laboratory product designed to facilitate the purification of long DNA fragments, such as those in the kilobase range. The kit utilizes magnetic beads to selectively bind and capture DNA molecules of the desired size, allowing for efficient separation and isolation from complex biological samples.

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Lab products found in correlation

Photoprobe biotin, by Vector Laboratories (1 mentions) Accuprep genomic dna extraction kit, by Bioneer (1 mentions) Hiseq 2000, by Illumina (1 mentions) Minelute reaction cleanup kit, by Qiagen (1 mentions) Puc19, by New England Biolabs (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Oct4 sc 5279, by Santa Cruz Biotechnology (1 mentions) Dnase, by Promega (1 mentions) Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions) Xhoi and xbai restriction enzymes, by New England Biolabs (1 mentions) Phusion hot start 2 high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Orbitrap fusion, by Thermo Fisher Scientific (1 mentions) Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

KilobaseBINDER Dynabeads Dynabeads

12 protocols using dynabeads kilobasebinder kit

Genomic DNA extraction and biotinylation

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Genomic DNA was extracted from 80% confluent dishes of MDA-MB-231 cells using the Accuprep Genomic DNA extraction Kit by Bioneer Corp (Daejeon, Republic of Korea) according to the manufacturer’s instructions. DNA was digested with Sac II, precipitated, and suspended in TE buffer and biotinylated by thermal coupling with photoprobe biotin (Vector Laboratories, Burlingame, CA, USA) per manufacturer’s instructions. Unlabeled biotin was removed and DNA was precipitated and resuspended in TE.
biotinylated DNA was pulled down by Dynabeads M-280 streptavidin using Dynabeads kilobaseBINDER kit (Invitrogen, Carlsbad, CA, USA) per manufacturer’s instructions. Briefly, the beads were incubated with DNA at room temperature with constant vertical shaking, washed and incubated with protein extract.

Nangia-Makker P., Shekhar M.P., Hogan V., Balan V, & Raz A. (2022). MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis. Oncotarget, 13, 534-550.

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Genome-wide Tn insertion mapping

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To increase the coverage of Tn insertion tags, 300 ng of purified DNA was digested with three restriction enzymes (DpnII, HaeIII, MspI), and then ligated with the corresponding DNA linkers. Ligation mixture were pooled and further digested with XbaI and KpnI to remove detection of Tn localized at donor site. Digested products were cleaned with MinElute Reaction Cleanup kit (28204, Qiagen), and the entire elute was used in primary PCR reactions with primers specific to Tn and linker sequences. The Tn-specific primer was biotinylated at 5′ end, which allowed enrichment of the PCR products by using the Dynabeads kilobaseBINDER kit (601-01, Invitrogen). PCR products were retrieved by incubation in 5 μl of 0.1 M NaOH for 10–20 min and 2 μl of it was further amplified with nested primers in secondary PCR. The nest PCR primers contained adaptor sequences, with which the sequencing library was constructed directly from purified secondary PCR products. Solexa sequencing was carried out on HiSeq 2000 (Illumina) at the Tufts Genomics Core. Sequences of PCR primers were listed in Supplementary Table 7. Raw and processed sequencing data will be available upon request.

Sun J., Ramos A., Chapman B., Johnnidis J.B., Le L., Ho Y.J., Klein A., Hofmann O, & Camargo F.D. (2014). Clonal dynamics of native haematopoiesis. Nature, 514(7522), 322-327.

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Biotinylation and Immobilization of Lamin B2 DNA

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A 2-kb fragment (lamin B2) corresponding to positions 2500–4500 bp of the human lamin B2 origin (GenBank^TM accession number M94363) was synthesized by PCR using the sense primer MW162 (5′-CGGGATCCTGCAGCTCAAGTCTTAAAGAC-3′), and the antisense primer MW163 (5′-GGGGTACCGGACTACAACTCCCACACGAC-3′), and subcloned into pUC19 (New England BioLabs).
The pUC19-lamin-B2 plasmids were biotinylated using PHOTOPROBE long-arm biotin (Vector Laboratories) according to the manufacturer's instruction. To generate biotinylated linear lamin B2 DNA, the 2-kb fragment was amplified by PCR with the primer MW162 labeled at the 5′ end with biotin. The PCR product was subjected to agarose gel electrophoresis, after which the corresponding band was excised and the lamin B2 fragment was purified with QiaQuick gel extraction kit (Qiagen) following the manufacturer's instruction.
The immobilization of biotinylated DNA to magnetic beads was carried out using the Dynabeads kilobaseBINDER kit (Invitrogen) according to the manufacturer's instruction. The amount of DNA bound to the beads was estimated by subtracting the amount of DNA in the flow-through after binding from the amount that was put in the reaction. Control beads without DNA underwent the same coupling procedure in the absence of DNA.

Wu M., Lu W., Santos R.E., Frattini M.G, & Kelly T.J. (2014). Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes. The Journal of Biological Chemistry, 289(44), 30810-30821.

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Coronin-1 Protein Interactome Identification

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DNA pull down assay was performed to find out protein interaction with coronin-1. The 5′-flanking fragment of coronin-1 was labeled with biotin by PCR amplification using 5′-terminal biotinylated forward primers (Table 1). The gene was bound to streptavidin magnetic beads using the Dynabeads KilobaseBINDER Kit following the manufacturer's protocol (Invitrogen). The nuclear proteins were incubated with biotinylated DNA—Dynabeads complex on a rotating shaker at 4°C overnight. The supernatant was removed and the complex was washed three times with cold PBS following the incubation. After the last washing, a pull down solution was resuspended with eluent at 70°C for 3 min to break the binding of streptavidin and biotin. The eluted DNA protein from the beads that did not contain the biotinylated DNA probe was used as the control, and Western blotting was performed to identify the specific protein.

Yang Z., Chen Y., Zhang Q., Chen X, & Deng Z. (2021). Major Outer Membrane Protein from Legionella pneumophila Inhibits Phagocytosis but Enhances Chemotaxis of RAW 264.7 Macrophages by Regulating the FOXO1/Coronin-1 Axis. Journal of Immunology Research, 2021, 9409777.

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Isolation and Analysis of Oct4-Binding Proteins

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A vector containing mouse Oct4 promoter was constructed. The DNA vector was treated with the appropriate restriction enzyme, and only the mouse Oct4 promoter was purified. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme. Biotinylated DNA was attached to streptavidin with Dynabeads kilobaseBINDER kit (60101)(Invitrogen, USA). The DNA was then incubated with the mESC protein extract. To remove the unbound proteins, beads were washed with washing buffer and magnets. Magnets pull streptavidin-Dynabeads, which were attached to biotinylated DNA with bound proteins. The last step was the elution of the proteins from the DNA and the protein analysis. After treatment with DNase (M6101)(Promega, USA) to eliminate DNA, we carefully took the supernatant. Finally, eluted proteins were separated using SDS-PAGE gel electrophoresis, and analyzed with MALDI-TOF and western blotting with Oct4 (sc-5279)(Santa Cruz, USA) and MBD2 (ab38646) (Abcam, UK) antibodies.

Kwon Y.W., Ahn H.S., Park J.Y., Yang H.M., Cho H.J, & Kim H.S. (2018). Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4. BMB Reports, 51(5), 242-248.

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Keratin-19 RNA Pulldown Proteomics

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An 18 nt RNA oligonucleotide biotinylated at the 5′ end was purchased from Integrated DNA Technologies. The oligonucleotide sequence matched the first 18 bases of the sense strand of our 1.56-kb keratin-19 fragment. The oligonucleotide was annealed to the antisense strand of our keratin-19 RNA fragment to produce the biotinylated ssRNA construct. The dsRNA construct was produced by also annealing the sense strand of the keratin-19 RNA fragment (minus the first 18 bases). Our biotinylated ssRNA and dsRNA constructs were coupled to streptavidin-coated magnetic beads using the Dynabeads KilobaseBINDER Kit (Invitrogen). The RNA-coupled beads were incubated in Xenopus egg extract for 90 min at 20°C. The beads were washed with a buffer containing 100 mM HEPES, pH 7.7, 1 mM MgCl₂, 100 mM KCl, and 50 mM sucrose. Proteins were eluted in a sample buffer containing 0.2% SDS + 40 mM DTT. Eluted samples were run on a 7% polyacrylamide gel. Bands of interest were excised and identified by tandem mass spectrometry (Taplin Mass Spectrometry Facility, Harvard Medical School).

Schweidenback C.T., Emerman A.B., Jambhekar A, & Blower M.D. (2015). Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis. RNA, 21(2), 279-295.

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Biotinylated cDNA Purification using Streptavidin Beads

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Biotinylated cDNAs were purified using streptavidin-coated magnetic beads (11205D, Dynabeads M-280 Streptavidin, Life Technologies) and the Dynabeads kilobaseBINDER Kit (60101, Life Technologies). For each RT reaction, a volume of 10 μl of magnetic beads was pre-equilibrated in 40 μl of Binding Solution. Once pre-equilibrated, 20 μl of the RT reaction was added to each aliquot of beads and incubated for 1.5 hr at room temperature on a rotator. Tubes were then placed on a DynaMag-96 Side Magnetic Particle Concentrator (123.31D, Life Technologies) to immobilize the beads in complex with the biotinylated cDNAs and the supernatant was removed. Each tube was then washed 3 times in 1X Washing Buffer and resuspended in the appropriate volume of PCR master mix, as indicated below.

Haist K., Ziegler C, & Botten J. (2015). Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs. PLoS ONE, 10(5), e0120043.

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Biotinylated DNA Enrichment and Detection

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The biotin incorporation assay was performed as described (Radecke et al. 2006b (link)) with minor modifications. The EGFP reporter cells were transfected with internal biotin-labeled ODNs (BFP_S90_Biotin) and flow-sorted as described above. The genomic DNA from 1 × 10⁵ BFP-positive cells was prepared using a Puregene Cell kit (Gentra) with a 60-min centrifugation step after isopropanol precipitation. The genomic DNA was digested to completion with XhoI and XbaI restriction enzymes (New England Biolabs). The biotinylated DNA fragments were isolated using a Dynabeads Kilobase BINDER kit (Life Technologies) according to the manufacturer's protocol, except that all reagents were supplemented with 0.1% BSA, and two additional washes using 0.1 M NaOH and 0.05 M NaCl at room temperature and one more rinse with 95°C water were performed to remove the noncovalently linked genomic fragments. The biotinylated genomic fragments were detected with a 40-cycle PCR using Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Scientific). The genomic DNA preparation prior to Dynabeads purification was used as control in a 25-cycle reaction.

Kan Y., Ruis B., Takasugi T, & Hendrickson E.A. (2017). Mechanisms of precise genome editing using oligonucleotide donors. Genome Research, 27(7), 1099-1111.

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Kinetics of PCNA Loading Assay

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We determined the kinetics of PCNA loading by a bead-based PCNA loading assay. ssM13 DNA (7 kbp; New England BioLabs) was coupled to streptavidin beads (ThermoFisher Dynabeads kilobaseBINDER Kit, 60101) overnight at room temperature using biotinylated oligos following manufacturer’s recommendations. Beads were resuspended in autoclaved water, and coupling efficiency was determined by PCR. Loading reaction occurred in a 1× Loading Buffer (50 mM Hepes, 4% glycerol, 0.01% NP-40, 250 mM Potassium Glutamate, 5 mM MgCl and 0.5 mM TCEP) with ∼50 ng of ssM13, 1 μM PCNA, 150 nM RFC, and 2 mM of various nucleotides. For elevated temperature experiments, PCNA was preincubated at 42 °C for 24 h prior to loading. Loading reactions were conducted at 25 °C for times indicated and quenched by adding 25 mM EDTA and chilling samples on ice. Following two washes with Loading buffer, we eluted all bound protein with Laemmli buffer. PCNA was detected by Western blot using an anti-PCNA antibody (Abcam, ab29) and quantified by ImageJ (67 ).

Magrino J., Munford V., Martins D.J., Homma T.K., Page B., Gaubitz C., Freire B.L., Lerario A.M., Vilar J.B., Amorin A., Leão E.K., Kok F., Menck C.F., Jorge A.A, & Kelch B.A. (2023). A thermosensitive PCNA allele underlies an ataxia-telangiectasia-like disorder. The Journal of Biological Chemistry, 299(5), 104656.

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Fabrication of Fluorescent DNA Dosimeter

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Fabrication of the dosimeter and details has been previously reported^{18 (link)}. Briefly, a polymerization chain reaction (PCR) was first used to combine oligonucleotide-attached fluorescein amidite (oligo-FAM) and biotin-labeled oligonucleotides with a pRS-316 vector as the template. The result is 4 kbp double stranded DNA with FAM on one end and biotin on the other on the other end. Gel electrophoresis was then used to verify strand length. After confirmation, these strands are then attached to streptavidin-coated magnetic MyOne T1 Dynabeads (Thermo Fisher Scientific, Waltham, MA) using the recommended binding buffer from the Dynabeads kilobase BINDER Kit (Thermo Fisher Scientific, Waltham, MA). After completing the attachment, unattached strands are finally removed from the samples. The resulting dosimeter can be seen in Figure 1, and the samples can be stored until they are used.

Bui B., McConnell K., Obeidat M., Saenz D., Papanikolaou N., Shim E.Y, & Kirby N. (2020). DNA dosimeter measurements of beam profile using a novel simultaneous processing technique. Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine, 165, 109316.

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