Chip dna clean concentrator

Manufactured by Zymo Research

The ChIP DNA Clean & Concentrator is a product designed to purify and concentrate ChIP (Chromatin Immunoprecipitation) DNA samples. It allows for the removal of contaminants and the concentration of the DNA samples, which is essential for downstream applications such as qPCR, sequencing, or further analysis.

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30 protocols using chip dna clean concentrator

ChIP-qRT-PCR Analysis of Protein-DNA Interactions

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ChIP-qRT-PCR assay was performed as previously described58 (link). DNA fragments derived from decross-linked protein-DNA complex were extracted and purified by ChIP DNA Clean & Concentrator (Zymo Research). Gene specific primer sets for qRT-PCR analysis are summarized in Supplementary Table S4.

Li H.K., Mai R.T., Huang H.D., Chou C.H., Chang Y.A., Chang Y.W., You L.R., Chen C.M, & Lee Y.H. (2016). DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma. Scientific Reports, 6, 28637.

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ChIP-qPCR Analysis of HY5 Binding

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Four-day-old seedlings of HY5pro:HY5-YFP in hy5-1 (in Ler background) and Ler were grown at 22 °C in the dark and transferred to light incubators for 4 h or not before harvested. ChIPs were carried out based on our established MOBE-ChIP method^{36 (link),41 (link),67 (link),68 (link)}. Briefly, around 12 g of samples were harvested and divided into small aliquots. Chromatin fragmentation was carried out on a Bioruptor^® sonicator (Diagenode) with the following settings: 12 high-intensity cycles of 30 s “on” and 30 s “off” at 4 °C. Immunoprecipitation was carried out at 4 °C overnight on a rotating platform using GFP-TRAP (ChromoTek, gtma-10). Reverse cross-linking of the immunoprecipitated complex was carried out at 65 °C for 7 h and purified by the ChIP DNA Clean & Concentrator (Zymo, D5201). Subsequent quantitative PCR reactions were performed with LUNA^® Universal qPCR Master Mix (New England Biolabs, M3003X) on a CFX96 Real-Time PCR detection system (Bio-Rad) using primers specific to the promoter of STOMAGEN or other specified regions (Supplementary Table 1) (IR1^{69 (link)}). Signals from the ChIPed DNA were normalized to their input DNA.

Wang S., Zhou Z., Rahiman R., Lee G.S., Yeo Y.K., Yang X, & Lau O.S. (2021). Light regulates stomatal development by modulating paracrine signaling from inner tissues. Nature Communications, 12, 3403.

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Chromatin Immunoprecipitation Assay for Transcription Factor Binding

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The ChIP assay was performed as previously described [41 ]. Briefly, the protein-DNA complexes were crosslinked by incubating the cells with 1% formaldehyde solution for 10 min at room temperature (RT). Then, the formaldehyde was quenched by 125 mm glycine for 5 min at RT. Cells were then lysed in a cold lysis buffer (1% SDS, 10 mmol l⁻¹ Tris-HCl, pH 8.0, 10 mmol l⁻¹ NaCl, 3 mmol l⁻¹ MgCl₂ and 0.5% NP-40) containing a protease inhibitor cocktail (Thermo Fisher Scientific). The samples were then sonicated to shear chromatin to an average length of about 1 kb. After centrifugation at 12 000×g for 10 min, the supernatant was pre-cleaned with protein A beads (Thermo Fisher Scientific) and then incubated with antibodies against RAR, retinoid X receptor, or normal rabbit IgG overnight at 4 °C. Then, the antibody-chromatin complex was precipitated with protein A beads, further treated with RNaseA and then proteinase K for 2 h to remove RNA and protein respectively. DNA was purified with ChIP DNA Clean & Concentrator (Zymo Research). The putative RAREs in the Vegfa and Prdm16 promoters were predicted (Supplementary Tables S2 and S5) using the JASPAR database (http://jaspardev.genereg.net/). Primers covering these sites are listed in Supplementary Tables S3 and S6.

Wang B., Fu X., Liang X., Deavila J.M., Wang Z., Zhao L., Tian Q., Zhao J., Gomez N.A., Trombetta S.C., Zhu M.J, & Du M. (2017). Retinoic acid induces white adipose tissue browning by increasing adipose vascularity and inducing beige adipogenesis of PDGFRα+ adipose progenitors. Cell Discovery, 3, 17036-.

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ChIP-seq Protocol for H3K9ac Profiling

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Twenty-five million CCRF-CEM parental and SLFN11-del cells were treated or untreated with CPT (100 nM) for 4 hours. ChIP assay was done by following the instruction manual of ChIP-IT Express (Active Motif). Briefly, cells were fixed with 1% formaldehyde in medium for 10 min at room temperature. Fixation was stopped with x1 glycine/PBS. Cells were lysed with lysis buffer with proteinase inhibitor cocktail and PMSF, and homogenized 60 times by small tight homogenizer. Cell pellets were re-suspended with sharing buffer, and sonicated by the following settings: pulse 20 s pulse, 40 s pause, amplitude 25%, repeat 5 times (QSONICA Sonicator, ultrasonic processor). Five %/volume of each sample was saved as input. The left of the supernatant was incubated with 2 μg antibody (H3K9ac or normal rabbit IgG) and protein G beads for overnight at 4°C. After reversing cross-link and proteinase K treatment, immunoprecipitated DNA was purified with ChIP DNA Clean & concentrator (ZYMO Research) according to the manual. Quantitative PCR was done using FastStart Universal SYBR Green Master (Roche) and ABI PRISM 7900TH.

Murai J., Tang S.W., Leo E., Baechler S.A., Redon C.E., Zhang H., Al Abo M., Rajapakse V.N., Nakamura E., Miller Jenkins L.M., Aladjem M.I, & Pommier Y. (2018). SLFN11 blocks stressed replication forks independently of ATR. Molecular cell, 69(3), 371-384.e6.

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ATAC-seq Library Preparation Protocol

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ATAC-seq libraries were prepared as previously described (Hoeksema et al., 2021 (link)). In brief, 5 × 10⁵ cells were lysed at room temperature in 50 µl ATAC lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and 2.5 µl DNA Tagmentation Enzyme mix (Nextera DNA Library Preparation Kit, Illumina) was added. The mixture was incubated at 37°C for 30 min and subsequently purified using the ChIP DNA Clean & Concentrator kit (Zymo Research) as described by the manufacturer. DNA was amplified using the Nextera Primer Ad1 and a unique Ad2.n barcoding primers using NEBNext High-Fidelity 2× PCR MM for 8–14 cycles. PCRs were size selected using TBE gels for 175–350 bp and DNA eluted using gel diffusion buffer (500 mM ammonium acetate, pH 8.0, 0.1% SDS, 1 mM EDTA, 10 mM magnesium acetate) and purified using ChIP DNA Clean & Concentrator (Zymo Research). Samples were quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 75 bp single-end sequenced on HiSeq 4000 (Illumina).

Shen Z., Li R.Z., Prohaska T.A., Hoeksema M.A., Spann N.J., Tao J., Fonseca G.J., Le T., Stolze L.K., Sakai M., Romanoski C.E, & Glass C.K. (2022). Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs. eLife, 11, e70878.

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ATAC-seq Protocol for Chromatin Profiling

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Approximately 80k cells were lysed in 50 µl room temperature ATAC lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), 2.5 µL DNA Tagmentation Enzyme mix (Nextera DNA Library Preparation Kit, Illumina) was added. The mixture was incubated at 37°C for 30 minutes and subsequently purified using the ChIP DNA purification kit (Zymo Research) as described by the manufacturer. DNA was amplified using the Nextera Primer Ad1 and a unique Ad2.n barcoding primers using NEBNext High-Fidelity 2X PCR MM for 8-14 cycles. PCR reactions were size selected using TBE gels for 175 -350 bp and DNA eluted using gel diffusion buffer (500 mM ammonium acetate, pH 8.0, 0.1% SDS, 1 mM EDTA, 10 mM magnesium acetate) and purified using ChIP DNA Clean & Concentrator (Zymo Research). Samples were quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 75bp single-end sequenced on HiSeq 4000 or NextSeq 500 (Illumina).

Hoeksema M.A., Shen Z., Holtman I.R., Zheng A., Spann N.J., Cobo I., Gymrek M., & Glass C.K. (2020). Mechanisms underlying divergent responses of genetically distinct macrophages to IL-4.

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ChIP-qPCR Analysis of Histone Modifications

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Antibodies against H3K4me1 (39298, Active Motif), H3K27ac (39134, Active Motif), and histone H3 (MABI0301, Wako) were used. ChIP assays were performed with DT40 cells (2 × 10⁶) and each antibody (3.5 µl for H3K4me1 or 2 µg for the others) as described previously.¹³ (link) DNA purified using ChIP DNA Clean & Concentrator (Zymo Research) was used as template for real-time PCR with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7900HT Fast Real-Time PCR System. Primers used in this experiment are shown in Supplementary Table S1.

Fujita T., Kitaura F., Yuno M., Suzuki Y., Sugano S, & Fujii H. (2017). Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 24(5), 537-548.

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ChIP-Seq with Tagmentation Library Prep

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Cells were cultured to 80% confluency and were cross-linked using 1% formaldehyde by rocking for 10 min at room temperature and then quenched with 0.125 M glycine. Cross-linked cells were washed in PBS and collected using a scrapper. Libraries were obtained following chromatin immunoprecipitation (ChIP)-tagmentation protocol described elsewhere.^{15 (link)} Briefly, a standard ChIP protocol was followed using the ChIP-IT Express Chromatin Immunoprecipitation Kit (Active Motif, 53008) that included cell lysis, sonication of chromatin (Covaris S220 ultrasonicator), and immunoprecipitation using antibodies bound to beads. At this step, the sequencing adapters were introduced in a single step by tagmentation (Nextera Tag DNA Enzyme) of bead-bound chromatin. Following tagmentation, the kit protocol was resumed with additional bead washes, tagmented ChIP-DNA elution, reverse cross-linking at 65°C overnight, and DNA purification (ChIP DNA Clean & Concentrator, Zymo Research). Library preparation was performed using custom Nextera primers as described for ATAC-seq. Library cluster generation and single-end sequencing was performed on the Illumina platform. Reads were aligned with BWA version 0.7.12^{16 (link)} to human genome reference of build Hg19, and duplicates were marked with Picard version 1.92 (http://broadinstitute.github.io/picard).

Slemmons K.K., Mukherjee S., Meltzer P., Purcell J.W, & Helman L.J. (2020). LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies. Pediatric blood & cancer, 68(2), e28771.

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ATAC-Seq Library Preparation Protocol

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Approximately 80K cells were lysed in 50-μl room temperature ATAC lysis buffer [10 mM tris-HCl, (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630], 2.5-μl DNA Tagmentation Enzyme mix (Nextera DNA Library Preparation Kit, Illumina) was added. The mixture was incubated at 37°C for 30 min and subsequently purified using the ChIP DNA purification kit (Zymo Research) as described by the manufacturer. DNA was amplified using the Nextera Primer Ad1 and a unique Ad2.n barcoding primer using NEBNext High-Fidelity 2× PCR MM for 8 to 14 cycles. PCRs were size-selected using TBE gels for 175 to 350 bp, and DNA was eluted using gel diffusion buffer [500 mM ammonium acetate (pH 8.0), 0.1% SDS, 1 mM EDTA, and 10 mM magnesium acetate] and purified using ChIP DNA Clean & Concentrator (Zymo Research). Samples were quantified by the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 75-bp single-end–sequenced on HiSeq 4000 or NextSeq 500 (Illumina).

Hoeksema M.A., Shen Z., Holtman I.R., Zheng A., Spann N.J., Cobo I., Gymrek M, & Glass C.K. (2021). Mechanisms underlying divergent responses of genetically distinct macrophages to IL-4. Science Advances, 7(25), eabf9808.

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Chromatin Immunoprecipitation of pSMAD3 Binding to COL1A2 Promoter

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NHDF lines were incubated with control or PTP4A1 ASO, serum-starved for 24 h and stimulated with 20 ng/ml human TGFβ1 or vehicle as control. Then cells were fixed in 1% formaldehyde for 15 min at RT. After sonication, chromatin was immunoprecipitated with the ChIP assay kit from Millipore using a rabbit anti-pSMAD3 (S423/425) antibody overnight at 4 °C. The eluted DNA was purified with ChIP DNA Clean & Concentrator from Zymo Research (Irvine, CA) and used for COL1A2 promoter qPCR. 10% input for each condition was used for normalization. Human COL1A2 promoter primers^{54 (link)}, 5′-TCTGCCCATGTCGGGGCT-3′ (forward) and 5′-TGCCTCCAAAAGGGCCTCC-3′ (reverse), were purchased from IDT.

Sacchetti C., Bai Y., Stanford S.M., Di Benedetto P., Cipriani P., Santelli E., Piera-Velazquez S., Chernitskiy V., Kiosses W.B., Ceponis A., Kaestner K.H., Boin F., Jimenez S.A., Giacomelli R., Zhang Z.Y, & Bottini N. (2017). PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis. Nature Communications, 8, 1060.

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