Poly di dc

Manufactured by Merck Group

Sourced in United States, Germany

Poly(dI-dC) is a synthetic DNA polymer composed of alternating deoxynucleotides of inosine and deoxycytidine. It is commonly used as a non-specific competitor DNA in various molecular biology applications, such as electrophoretic mobility shift assays (EMSAs) and DNA-protein binding studies.

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Lab products found in correlation

T4 polynucleotide kinase, by New England Biolabs (5 mentions) γ 32p atp, by PerkinElmer (5 mentions) Streptavidin agarose beads, by Merck Group (3 mentions) Irdye800 nhs esther, by LI COR (2 mentions) Qiaquick column, by Qiagen (2 mentions) T7 sequenase 2.0 dna sequencing kit, by Cytiva (2 mentions) γ 32p datp, by PerkinElmer (2 mentions) Nylon membrane, by Cytiva (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Criterion xt 4 12 bis tris gel, by Bio-Rad (1 mentions) Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions) Dna loading dye, by Thermo Fisher Scientific (1 mentions) Typhoon 8600 imager, by GE Healthcare (1 mentions) Bl21 de3 plyss, by Promega (1 mentions) Yeast trna, by Thermo Fisher Scientific (1 mentions) Exposed phosphor screens, by GE Healthcare (1 mentions) Typhoon fla plate reader, by GE Healthcare (1 mentions) Salmon sperm dna, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Imagequant tl, by GE Healthcare (1 mentions)

58 protocols using poly di dc

Transcriptional Regulation Binding Assay

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EMSAs were conducted as previously described (24 (link)). Briefly, a 646-bp csgD gene promoter including 237 nucleotides in the open reading frame was amplified from S11923-3 and purified with an AxyPrep DNA Gel Extraction Kit (Axygen), and the purified products were PCR labeled using FAM-modified primer. The binding reaction was conducted with non-specific competitor DNA (poly dI-dC, Sigma-Aldrich) in buffer (pH 7.4) containing 750 mM of NaCl, 0.5 mM of DTT, 0.5 mM of EDTA, and 50 mM of Tris at 25°C for 30 min. A total of 20 μL of each reaction system contained 4 μL 5 × binding buffer (Beyotime Biotechnology) and 200 ng of poly dI-dC (Sigma-Aldrich). The final mixtures including DNA fragments and RpoS protein were run on a 6% SDS-PAGE. Then the images were scanned and observed using a fluorescence imaging system (Typhoon FLA 9500, GE Healthcare).

Feng Z., El Hag M., Qin T., Du Y., Chen S, & Peng D. (2020). Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum. Frontiers in Veterinary Science, 7, 571361.

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Electrophoretic Mobility Shift Assay

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Electrophoretic mobility shift assay (EMSA) was performed as previously reported [73 (link)]. Complementary forward and reverse oligonucleotides (synthesized at IDT, Coralville, IA) were annealed to form dsDNA probes, wt-Ori-39 and mut-Ori-39 that had the STAT5BE mutated [71 (link)] (Fig 2B). The probes were 5’ end labeled with ³²P using [γ-³²P] ATP. Each 20 μl binding reaction contained 3 μg/ml of poly dI-dC (MilliporeSigma).

Ganaie S.S., Zou W., Xu P., Deng X., Kleiboeker S, & Qiu J. (2017). Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex. PLoS Pathogens, 13(5), e1006370.

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Transfection of ISD and poly-dI/dC

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Two microgram of ISD (Invivogen) or 1 μg each of ISD and poly-dI/dC (Millipore-Sigma) was transfected into cells using lipofectamine 2000 (Invitrogen) per manufacturer’s protocol. Cells were incubated for 6 h and harvested for RNA analyses or fractionation. Mock-transfected controls received lipofectamine/Optimem mixture only.

Lazarchuk P., Nguyen V.N., Brunon S., Pavlova M.N, & Sidorova J.M. (2023). Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells. Frontiers in Molecular Biosciences, 9, 1048726.

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Protein-DNA Interaction Profiling

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Streptavidin magnetic beads (200 μg; Dynabeads M-280 Streptavidin (#112-06D); Invitrogen, Carlsbad, CA) were subjected to two rounds of blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 15 min followed by washing with PBS containing 1M NaCl and TE buffer. Biotinylated non-risk or risk oligonucleotides were bound to 100 μL of the BSA-blocked streptavidin beads by incubating for 30 min at room temperature in TE buffer, followed by washing with TE buffer. A biotinylated scrambled oligonucleotide served as a negative control (Supplementary Table 1). Fifty micrograms of nuclear extract were pre-cleared by incubating with 100 μL of the BSA-blocked beads in binding buffer (250 mM NaCl, 50 mM Tris Cl, 50% glycerol, 2.5 mM DTT, 2.5 mM EDTA, pH 7.6) containing 15 ng/μL poly dI:dC (#81349-500UG; Sigma-Aldrich, St. Louis, MO), 0.5 μg/mL BSA, and 0.1% NP40 for 30 min on ice. Pre-cleared nuclear extracts were incubated with the oligonucleotide-linked Streptavidin beads for 30 min in a 37 °C water bath. Samples were gently shaken every 5 min. Beads were subsequently washed three times with binding buffer containing 0.1% NP40. The proteins were eluted in 50 μL of 0.2% SDS sample buffer by boiling for 5 min then resolved on a Criterion XT 4%–12% Bis-Tris gel (#3450124; BioRad, Hercules, CA) followed by western blotting.

Pasula S., Tessneer K.L., Fu Y., Gopalakrishnan J., Pelikan R.C., Kelly J.A., Wiley G.B., Wiley M.M, & Gaffney P.M. (2020). Risk Variants with Opposing Functional Effects Result in Hypomorphic Expression of TNIP1 and Other Genes within a 3D Chromatin Network. Arthritis & rheumatology (Hoboken, N.J.), 72(5), 780-790.

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ParB-Mediated parS Binding Assay

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To test the ability of ParB-His₆ to bind the parS motif, 0.01 µM of a Cy3-labeled double-stranded DNA oligonucleotide containing either the wild-type (5′-TGTTTCACGTGAAACA-3′) or a mutated (5′-TGCCTCACGTGAAACA-3′) parS sequence were incubated with 0.05 µg µl⁻¹ poly(dI-dC) (Sigma Aldrich, Germany) and varying concentrations of ParB (0–0.6 µM) in buffer B6 (50 mM HEPES/NaOH, pH 7.2, 50 mM NaCl, 5 mM MgCl_2, 0.1 mM EDTA, 10% glycerol) for 30 min at 28 °C. 20 µl samples were mixed with 6× DNA loading dye (Fermentas, Germany) and loaded on a 6% non-denaturing polyacrylamide gel, which was run for 50 min at a constant voltage of 120 V and 4 °C in 1× TBE buffer. Signals were detected with a Typhoon 8600 imager (GE Healthcare).

Jung A., Raßbach A., Pulpetta R.L., van Teeseling M.C., Heinrich K., Sobetzko P., Serrania J., Becker A, & Thanbichler M. (2019). Two-step chromosome segregation in the stalked budding bacterium Hyphomonas neptunium. Nature Communications, 10, 3290.

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Analyzing Nuclear Protein-DNA Interactions in Lung Cancer

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Nuclear extracts of lung cancer cell lines H1299 and A549 were prepared using the method described by Edmead et al15 (link). The sequences of the biotin-labeled double-stranded oligonucleotide probes of rs6486433, rs3763869 and rs3763868 are listed in Table S1. EMSA was carried out according to the manufacturer’s protocol (EMSA Kit ES009, Beyotime, China). Briefly, 10 μg nuclear extract were incubated in 20 μl binding reaction buffer (10 mM Tris-HCl, pH7.5, 50 mM KCl, 1 mM DTT, 10% glycerol) with the 2.5 nM biotin-labeled probe and 0.5 μg poly dI:dC (Sigma) for 30 min at room temperature. DNA–protein complexes were subjected to electrophoresis on 5% polyacrylamide gel, transferred to nylon membrane, and visualized by chemiluminescence imaging.

Liu D., Wu C., Jiao Y., Hou L., Lu D., Zheng H., Chen C., Qian J., Fei K, & Su B. (2015). WEE1 kinase polymorphism as a predictive biomarker for efficacy of platinum-gemcitabine doublet chemotherapy in advanced non-small cell lung cancer patients. Scientific Reports, 5, 11114.

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HNF4 Transcription Factor Binding Assay

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The WT (5′-CAGTGAACTTAGGTCCTGATT-3′) and mutant form (5′-CAGTGTTGTTAGGTCCTGATT-3′) of the 5′ biotinylated probes containing HNF4 responsive element were annealed with their complementary oligonucleotides in 20 μl of annealing buffer (40 mM Tris-HCl pH7.5, 20 mM MgCl₂ and 50 mM NaCl) at 80 °C for 5 min. Nuclear extracts (300 μg) of transfected HuH7 cells were precleared with streptavidin-agarose resins (Sigma) and then incubated with 125 mg/ml poly(dI.dC) (Sigma) at 4 °C for 30 min in 300 μl binding buffer (18 mM HEPES pH 7.9, 40 mM KCl, 2 mM MgCl₂, 10 mM DTT and protease inhibitor). The mixtures were incubated with annealed biotinylated probe and 20 μl of streptavidin-agarose resins at room temperature for 1 hr. After extensive wash with binding buffer, the associated protein complexes were analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody.

Tsai T.Y., Wang W.T., Li H.K., Chen W.J., Tsai Y.H., Chao C.H, & Wu Lee Y.H. (2017). RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP. Scientific Reports, 7, 41452.

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Protein-DNA Interaction Profiling

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Four oligonucleotides containing biotin on the 5′-nucleotide of the sense strand were used in the pull-down assays. One microgram of each double-stranded oligonucleotide was incubated with 300 μg of nuclear protein extracts for 20 min at room temperature. Streptavidin-agarose beads (30 μl, Sigma-Aldrich) that had been pre-absorbed by poly(dI-dC) (Sigma-Aldrich) were added and incubated with the extract for 4 h at 4 °C. The protein-DNA-streptavidin-agarose complexes were pelleted by centrifugation and analyzed by Western blotting with Myc-tag antibody.

Wang R.H., Zhao T., Cui K., Hu G., Chen Q., Chen W., Wang X.W., Soto-Gutierrez A., Zhao K, & Deng C.X. (2016). Negative reciprocal regulation between Sirt1 and Per2 modulates the circadian clock and aging. Scientific Reports, 6, 28633.

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Characterization of Nuclear Protein Binding

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Nuclear proteins were prepared from MEL cells at differentiation day 2, as described previously (11 (link)). Recombinant GST-p66α proteins were obtained from the IPTG-induced BL21(DE3)pLysS (Promega L1195) strain of E. coli. EMSAs were carried out using DNA probes modified with [α³²P]-labels (Perkin Elmer). Equimolar amounts of complementary strands were mixed and heated to 95°C then gradually cooled to ambient temperature over a period no less than 4 hours to anneal probes. The probe DNA corresponded to the CP2c consensus binding sites in the mouse α-globin promoter: 5′-GAT CCC AAG TTT TAC TCG GTA GAG CAA GCA CAA ACC AGG-3′ (–156 to –124 from the start codon). For binding studies, double-stranded DNA probes were mixed with 2 μg of nuclear proteins and 1 μg of Poly dI-dC (Sigma Aldrich P4929) in binding buffer (100 mM KCl, 10 mM Tris–HCl, pH 7.9, 1 mM EDTA,1 mM DTT, 4% glycerol, 0.1% NP-40) and incubated at 37°C for 30 min. The reaction mixtures were separated on native 5% polyacrylamide gel for 1 h at 200 V and the dried gels were auto-radiographed. For supershift analysis, 2 μg polyclonal anti-p66α (Abcam ab87663) or polyclonal anti-CP2c (Cosmogenetec) antibody was added to the reaction mixture.

Kim M.Y., Kim J.S., Son S.H., Lim C.S., Eum H.Y., Ha D.H., Park M.A., Baek E.J., Ryu B.Y., Kang H.C., Uversky V.N, & Kim C.G. (2018). Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis. Nucleic Acids Research, 46(10), 4933-4949.

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DNA-Protein Interaction Profiling Protocol

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The biotinylated DNA fragments were incubated with streptavidin beads (S-1638; Sigma, St Louis, MO, USA) at 4°C overnight and washed three times with DAPA buffer (137 mM NaCl, 2.7 mM KCl, 7.7 mM NaH2PO4, 1.5 mM KH2PO4, 0.1% NP-40, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol). Subsequently, the DNA-conjugated beads were incubated with 2 mg of cell lysates and 10 μg of Poly dI-dC (Sigma; P4929) at 4oC overnight. Following the incubation, 30 μl of streptavidin-agarose beads (Millipore) was added to the reaction and incubated at 4°C for 1 h. The beads were then collected by centrifugation and washed four times with DAPA buffer containing 0.5% NP-40. The pulled down complexes were then resolved by 15% SDS–PAGE and analyzed by immunoblotting. The DNA probe sequences are listed in Supplementary Table S8.

Tsai P.H., Chien Y., Wang M.L., Hsu C.H., Laurent B., Chou S.J., Chang W.C., Chien C.S., Li H.Y., Lee H.C., Huo T.I., Hung J.H., Chen C.H, & Chiou S.H. (2019). Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network. Nucleic Acids Research, 47(19), 10115-10133.

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