recombinant dnase i  (Millipore)


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  • 99
    Name:
    DNase I recombinant
    Description:
    Double strand specific endonuclease that requires bivalent cations for maximal activity
    Catalog Number:
    04536282001
    Price:
    None
    Applications:
    . DNase I, recombinant, grade I is used for:Eliminating DNA during protein isolation procedures. Analysis of chromatin structure. Eliminating DNA during sample preparation
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    Structured Review

    Millipore recombinant dnase i
    Endothelial damage and complement activation by ANCA-induced NET generation. ( A ) HUVECs were coincubated with NETs isolated from either TNFα-primed human neutrophils without further stimulation (unstim), isotype mAb-stimulated (ctrl), or αMPO mAb-stimulated (αMPO) neutrophils. Permeability of the EC monolayer was measured after 1 h using albumin-FITC. NET degradation by DNase protected from endothelial damage. ( B ) Human neutrophil preincubation with Nec-1 protected from NET-induced endothelial damage. ( C ) NETs were coincubated in the absence (w/o) or presence of human serum and stained after PFA fixation with PI (DNA; red) and αC3d antibody (FITC; green). Unstimulated cells and isotype-stimulated neutrophils displayed no NETs and no C3d deposition, whereas αMPO mAb-stimulated cells coincubated with serum showed NET formation with strong C3d costaining. DNase pretreatment prevented C3d staining. A typical example is depicted. ( D ) NETs were coincubated with different human sera as indicated and stained after fixation with PI (DNA; red) and αC3d antibody (FITC; green). Isotype-stimulated neutrophils displayed no NETs and no C3d deposition; αMPO mAb-stimulated neutrophils displayed NET formation and—when coincubated with serum—C3d deposition. C3d deposition was not affected by C1q deficiency but strongly reduced in both C3-deficient (C3-def) and complement factor B-deficient (FB-def) sera. ( E ) αMPO mAb-induced NETs that were coincubated with human serum caused complement activation with C5a generation. The effect was prevented by NET degradation using DNase I. ( F ) Increased EC permeability by αMPO mAb-induced NETs is mediated by the alternative complement pathway, as both C3-deficient and complement factor B-deficient sera provided protection whereas C1q deficiency did not. Error bars indicate means ± SEM. Comparisons were made using  t  test or ANOVA; * P
    Double strand specific endonuclease that requires bivalent cations for maximal activity
    https://www.bioz.com/result/recombinant dnase i/product/Millipore
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    recombinant dnase i - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis"

    Article Title: Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1708247114

    Endothelial damage and complement activation by ANCA-induced NET generation. ( A ) HUVECs were coincubated with NETs isolated from either TNFα-primed human neutrophils without further stimulation (unstim), isotype mAb-stimulated (ctrl), or αMPO mAb-stimulated (αMPO) neutrophils. Permeability of the EC monolayer was measured after 1 h using albumin-FITC. NET degradation by DNase protected from endothelial damage. ( B ) Human neutrophil preincubation with Nec-1 protected from NET-induced endothelial damage. ( C ) NETs were coincubated in the absence (w/o) or presence of human serum and stained after PFA fixation with PI (DNA; red) and αC3d antibody (FITC; green). Unstimulated cells and isotype-stimulated neutrophils displayed no NETs and no C3d deposition, whereas αMPO mAb-stimulated cells coincubated with serum showed NET formation with strong C3d costaining. DNase pretreatment prevented C3d staining. A typical example is depicted. ( D ) NETs were coincubated with different human sera as indicated and stained after fixation with PI (DNA; red) and αC3d antibody (FITC; green). Isotype-stimulated neutrophils displayed no NETs and no C3d deposition; αMPO mAb-stimulated neutrophils displayed NET formation and—when coincubated with serum—C3d deposition. C3d deposition was not affected by C1q deficiency but strongly reduced in both C3-deficient (C3-def) and complement factor B-deficient (FB-def) sera. ( E ) αMPO mAb-induced NETs that were coincubated with human serum caused complement activation with C5a generation. The effect was prevented by NET degradation using DNase I. ( F ) Increased EC permeability by αMPO mAb-induced NETs is mediated by the alternative complement pathway, as both C3-deficient and complement factor B-deficient sera provided protection whereas C1q deficiency did not. Error bars indicate means ± SEM. Comparisons were made using  t  test or ANOVA; * P
    Figure Legend Snippet: Endothelial damage and complement activation by ANCA-induced NET generation. ( A ) HUVECs were coincubated with NETs isolated from either TNFα-primed human neutrophils without further stimulation (unstim), isotype mAb-stimulated (ctrl), or αMPO mAb-stimulated (αMPO) neutrophils. Permeability of the EC monolayer was measured after 1 h using albumin-FITC. NET degradation by DNase protected from endothelial damage. ( B ) Human neutrophil preincubation with Nec-1 protected from NET-induced endothelial damage. ( C ) NETs were coincubated in the absence (w/o) or presence of human serum and stained after PFA fixation with PI (DNA; red) and αC3d antibody (FITC; green). Unstimulated cells and isotype-stimulated neutrophils displayed no NETs and no C3d deposition, whereas αMPO mAb-stimulated cells coincubated with serum showed NET formation with strong C3d costaining. DNase pretreatment prevented C3d staining. A typical example is depicted. ( D ) NETs were coincubated with different human sera as indicated and stained after fixation with PI (DNA; red) and αC3d antibody (FITC; green). Isotype-stimulated neutrophils displayed no NETs and no C3d deposition; αMPO mAb-stimulated neutrophils displayed NET formation and—when coincubated with serum—C3d deposition. C3d deposition was not affected by C1q deficiency but strongly reduced in both C3-deficient (C3-def) and complement factor B-deficient (FB-def) sera. ( E ) αMPO mAb-induced NETs that were coincubated with human serum caused complement activation with C5a generation. The effect was prevented by NET degradation using DNase I. ( F ) Increased EC permeability by αMPO mAb-induced NETs is mediated by the alternative complement pathway, as both C3-deficient and complement factor B-deficient sera provided protection whereas C1q deficiency did not. Error bars indicate means ± SEM. Comparisons were made using t test or ANOVA; * P

    Techniques Used: Activation Assay, Isolation, Permeability, Staining

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    Clone Assay:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The coding region of OmBBD was amplified and cloned into the Gateway vector pDEST15 (Invitrogen), which is a GST tag plasmid, using the primers containing the recombination sites (Supplemental Table S1). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Centrifugation:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: For AAV purification by iodixanol density centrifugation, discontinuous gradients comprised of 15%, 25%, 40%, and 54% iodixanol layers were set up in Optiseal tubes (Beckman Coulter, 362185) using OptiPrep (Axis-Shield, AVS-1114542). .. DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195).

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: For expression of recombinant Sph2, the culture was incubated overnight at 16°C and harvested by centrifugation. .. To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Amplification:

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma). .. Quantitative RT-PCR amplification was performed with a SYBR green kit (TaKaRa, Dalian, China) and the CFX96 system (Bio-Rad, CA, USA).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The coding region of OmBBD was amplified and cloned into the Gateway vector pDEST15 (Invitrogen), which is a GST tag plasmid, using the primers containing the recombination sites (Supplemental Table S1). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). .. Primers for RT-qPCR (Supplementary Table ) were designed using NCBI Primer-BLAST and the primer amplification efficiency for each primer pair was between 1.9 and 2.1.

    Filtration:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: Vector preps were then buffer exchanged and concentrated into PBS with 0.001% or 0.00025% Tween 20 (Sigma-Aldrich, 9416) using Amicon filtration (EMD Millipore, UFC910024) at 3000 g . .. DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195).

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001). .. Protein was eluted in lysis buffer containing 200 mM Imidazole and then further purified via gel filtration (Superdex 200 HiLoad 16/600, GE Healthcare—28-9893-35) in CSF-XB buffer (0.01 M Hepes, 0.002 M MgCl, 0.0001 M CaCl, 0.004 M Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 10% w/v sucrose, pH 7.75) containing either 0.1 M KCl for extract assays or 0.5 M KCl for condensate assays.

    Mass Spectrometry:

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: Real-Time Quantitative RT-PCR Analysis Fourteen-day-old seedlings of wild-type, bzip19/23 mutant, bzip19/23 -OE19, bzip19/23 -OE23 and bzip19/23 -OE24 lines, grown in control or –Zn MS medium were harvested and immediately frozen in liquid nitrogen in pools of 5 seedlings per line and per Zn treatment × 3 different plates grown simultaneously and considered as biological replica. .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen).

    Positive Control:

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. The nucleus of all cells was stained with Hoechst 33342 (1:100, Sigma-Aldrich) and the nucleus of dead cells were counterstained with propidium iodide (1:100, Invitrogen) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield mounting medium (Vector laboratories).

    Quantitative RT-PCR:

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Paragraph title: Expression analysis for miR-like RNA using stem-loop RT-qPCR. ... Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma).

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: Paragraph title: Real-Time Quantitative RT-PCR Analysis ... The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen).

    SYBR Green Assay:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: .. DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195). .. To synthesize sgRNAs in vitro , a DNA template that contained a T7 promoter, a 20-nt guide sequence, and a sgRNA scaffold was generated by overlapping PCR.

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma). .. Quantitative RT-PCR amplification was performed with a SYBR green kit (TaKaRa, Dalian, China) and the CFX96 system (Bio-Rad, CA, USA).

    Incubation:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl. .. For the nucleic acid degradation assay, 15 μ g of total RNA, circular plasmid, digested linear plasmid, or genomic DNA prepared from leaves of O. minuta was incubated with the recombinant GST-OmBBD proteins for 2 h at 56°C.

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: The slides were rinsed twice with PBS and incubated with TUNEL reaction mixture for 1 h at 37°C within a humidified darkened container. .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: For expression of recombinant Sph2, the culture was incubated overnight at 16°C and harvested by centrifugation. .. To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Stripping Membranes:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes. .. These presented imaging, probe stripping, and hybridization protocols are repeated based on the barcoding scheme .

    Activity Assay:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl. .. For the nucleic acid degradation assay, 15 μ g of total RNA, circular plasmid, digested linear plasmid, or genomic DNA prepared from leaves of O. minuta was incubated with the recombinant GST-OmBBD proteins for 2 h at 56°C.

    Expressing:

    Article Title: Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships
    Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Pellets were lysed with 1x Bugbuster (Novagen) and DNase for 30 min at room temperature and then centrifuged at 4°C under high speed to collect the supernatant.

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Paragraph title: Expression analysis for miR-like RNA using stem-loop RT-qPCR. ... Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma).

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: Paragraph title: Protein constructs, expression, and purification ... For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The resulting in-frame fusion plasmid was transformed into Escherichia coli BL21 (DE3)-pLys, and expression in E. coli was then induced by the addition of 0.5 m m isopropyl- β - d -thiogalactoside at 30°C for 3 h. The recombinant OmBBD-GST proteins were purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and 10 m m reduced glutathione (Sigma) dissolved in 50 m m Tris-HCl (pH 8.0). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: Paragraph title: Recombinant protein expression and purification ... To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Transformation Assay:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: All constructs were transformed into Rosseta2 Escherichia coli cells and were grown in temperature-controlled incubators shaking at 200 r.p.m. .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The resulting in-frame fusion plasmid was transformed into Escherichia coli BL21 (DE3)-pLys, and expression in E. coli was then induced by the addition of 0.5 m m isopropyl- β - d -thiogalactoside at 30°C for 3 h. The recombinant OmBBD-GST proteins were purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and 10 m m reduced glutathione (Sigma) dissolved in 50 m m Tris-HCl (pH 8.0). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Hybridization:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: A piece of glass slide or parafilm was used to cover the top facet of the flow cell, preventing the imaging or hybridization buffer from evaporation. .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes.

    TUNEL Assay:

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: The slides were rinsed twice with PBS and incubated with TUNEL reaction mixture for 1 h at 37°C within a humidified darkened container. .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks.

    Flow Cytometry:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: A piece of glass slide or parafilm was used to cover the top facet of the flow cell, preventing the imaging or hybridization buffer from evaporation. .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes.

    RNA In Situ Hybridization:

    Article Title: Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner
    Article Snippet: Paragraph title: RNA in situ hybridization ... After treatment with RNase-free DNase I (04716728001, Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C, probes were hydrolyzed in hydrolysis buffer (40 mM NaHCO3 , 60 mM Na2 CO3 ) at 60°C for up to 30 min, depending on probe size.

    Protease Inhibitor:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001). ..

    Nick Translation:

    Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells
    Article Snippet: .. Probes were labeled for use in FISH by nick translation as follows: prior to nick translation, 1 μg DNA was treated with RNase (0.02 U) (Sigma), for 30 min at 37°C, nick translation was carried out at 16°C for 1 h in the following reaction mixture; 50 mM Tris-HCl, 5 mM MgCl2 , 2.5 μg BSA, 10 mM β-mercaptoethanol, 50 mM dAGC, 20 μM hapten/fluor [digoxigenin-11-dUTP (Sigma); Cy3 dUTP (GE Healthcare)], 15 U recombinant DNase1 (Sigma) and 10 U DNA polymerase I (NEB), made up to a final volume of 50 μL with H2 0. .. Imaging Equipment and Settings Widefield fluorescence imaging was performed at 20°C on a DeltaVision Elite system (Applied Precision) equipped with a 100x/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40), FITC (excitation 475/28; emission 525/45) and TRITC (excitation 542/27; emission 593/45) filters.

    Cell Culture:

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. Fragments from fresh tissue were cultured for 24h and 5 days such as control to evaluate the warming influence in survival and cell apoptosis after culture.

    Generated:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: This sequence was generated by interspacing nuclear localization-like sequences (e.g., KKRK) with inert a.a.’s G, S, A, as well as, oppositely charged E. The construct was designed to have a similar theoretical isoelectric point (pI) to the endogenous NT1-480aa of TPX2. .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001).

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). .. Primers for RT-qPCR (Supplementary Table ) were designed using NCBI Primer-BLAST and the primer amplification efficiency for each primer pair was between 1.9 and 2.1.

    Imaging:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes. .. The cells were subsequently hybridized by another probe set at 1 nM concentration for more than 12 hours at room temperate in the hybridization buffer.

    Sequencing:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: This sequence was generated by interspacing nuclear localization-like sequences (e.g., KKRK) with inert a.a.’s G, S, A, as well as, oppositely charged E. The construct was designed to have a similar theoretical isoelectric point (pI) to the endogenous NT1-480aa of TPX2. .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001).

    Recombinant:

    Article Title: Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships
    Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Pellets were lysed with 1x Bugbuster (Novagen) and DNase for 30 min at room temperature and then centrifuged at 4°C under high speed to collect the supernatant.

    Article Title: Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis
    Article Snippet: .. C1q-deficient and factor B-deficient sera were from Quidel, C3-deficient serum was from Sigma, propidium iodide, BSA-FITC, PMA, necrostatin-1, and recombinant DNase I were from Sigma-Aldrich, necrostatin-1s was from VWR, necrosulfonamide was from Millipore, and Sytox green was from Invitrogen. .. Human TNFα, murine TNFα, LPS (O26:B6), and Ficoll-Hypaque were from Sigma-Aldrich.

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: Paragraph title: Production and purification of recombinant AAV vectors ... DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195).

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: .. Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma). .. A stem-loop reverse transcription-quantitative PCR (RT-qPCR) method was used to quantitate miR-like RNA expression as in a previous study ( ).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The resulting in-frame fusion plasmid was transformed into Escherichia coli BL21 (DE3)-pLys, and expression in E. coli was then induced by the addition of 0.5 m m isopropyl- β - d -thiogalactoside at 30°C for 3 h. The recombinant OmBBD-GST proteins were purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and 10 m m reduced glutathione (Sigma) dissolved in 50 m m Tris-HCl (pH 8.0). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. The nucleus of all cells was stained with Hoechst 33342 (1:100, Sigma-Aldrich) and the nucleus of dead cells were counterstained with propidium iodide (1:100, Invitrogen) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield mounting medium (Vector laboratories).

    Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells
    Article Snippet: .. Probes were labeled for use in FISH by nick translation as follows: prior to nick translation, 1 μg DNA was treated with RNase (0.02 U) (Sigma), for 30 min at 37°C, nick translation was carried out at 16°C for 1 h in the following reaction mixture; 50 mM Tris-HCl, 5 mM MgCl2 , 2.5 μg BSA, 10 mM β-mercaptoethanol, 50 mM dAGC, 20 μM hapten/fluor [digoxigenin-11-dUTP (Sigma); Cy3 dUTP (GE Healthcare)], 15 U recombinant DNase1 (Sigma) and 10 U DNA polymerase I (NEB), made up to a final volume of 50 μL with H2 0. .. Imaging Equipment and Settings Widefield fluorescence imaging was performed at 20°C on a DeltaVision Elite system (Applied Precision) equipped with a 100x/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40), FITC (excitation 475/28; emission 525/45) and TRITC (excitation 542/27; emission 593/45) filters.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: Paragraph title: Recombinant protein expression and purification ... To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). .. Primers for RT-qPCR (Supplementary Table ) were designed using NCBI Primer-BLAST and the primer amplification efficiency for each primer pair was between 1.9 and 2.1.

    Mutagenesis:

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: Real-Time Quantitative RT-PCR Analysis Fourteen-day-old seedlings of wild-type, bzip19/23 mutant, bzip19/23 -OE19, bzip19/23 -OE23 and bzip19/23 -OE24 lines, grown in control or –Zn MS medium were harvested and immediately frozen in liquid nitrogen in pools of 5 seedlings per line and per Zn treatment × 3 different plates grown simultaneously and considered as biological replica. .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen).

    Labeling:

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. The nucleus of all cells was stained with Hoechst 33342 (1:100, Sigma-Aldrich) and the nucleus of dead cells were counterstained with propidium iodide (1:100, Invitrogen) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield mounting medium (Vector laboratories).

    Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells
    Article Snippet: .. Probes were labeled for use in FISH by nick translation as follows: prior to nick translation, 1 μg DNA was treated with RNase (0.02 U) (Sigma), for 30 min at 37°C, nick translation was carried out at 16°C for 1 h in the following reaction mixture; 50 mM Tris-HCl, 5 mM MgCl2 , 2.5 μg BSA, 10 mM β-mercaptoethanol, 50 mM dAGC, 20 μM hapten/fluor [digoxigenin-11-dUTP (Sigma); Cy3 dUTP (GE Healthcare)], 15 U recombinant DNase1 (Sigma) and 10 U DNA polymerase I (NEB), made up to a final volume of 50 μL with H2 0. .. Imaging Equipment and Settings Widefield fluorescence imaging was performed at 20°C on a DeltaVision Elite system (Applied Precision) equipped with a 100x/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40), FITC (excitation 475/28; emission 525/45) and TRITC (excitation 542/27; emission 593/45) filters.

    Article Title: Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner
    Article Snippet: Restriction digest and in vitro transcription were done according to manufacturer’s instructions, with DIG RNA Labeling Mix (11277073910, Sigma-Aldrich, St. Louis, MO). .. After treatment with RNase-free DNase I (04716728001, Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C, probes were hydrolyzed in hydrolysis buffer (40 mM NaHCO3 , 60 mM Na2 CO3 ) at 60°C for up to 30 min, depending on probe size.

    Purification:

    Article Title: Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships
    Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Pellets were lysed with 1x Bugbuster (Novagen) and DNase for 30 min at room temperature and then centrifuged at 4°C under high speed to collect the supernatant.

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: Paragraph title: Production and purification of recombinant AAV vectors ... DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195).

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: Paragraph title: Protein constructs, expression, and purification ... For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The resulting in-frame fusion plasmid was transformed into Escherichia coli BL21 (DE3)-pLys, and expression in E. coli was then induced by the addition of 0.5 m m isopropyl- β - d -thiogalactoside at 30°C for 3 h. The recombinant OmBBD-GST proteins were purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and 10 m m reduced glutathione (Sigma) dissolved in 50 m m Tris-HCl (pH 8.0). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: Paragraph title: Recombinant protein expression and purification ... To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Polymerase Chain Reaction:

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma). .. A stem-loop reverse transcription-quantitative PCR (RT-qPCR) method was used to quantitate miR-like RNA expression as in a previous study ( ).

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). .. RT-qPCR was performed with a LightCycler 96 Real-Time PCR Systtem (Roche Diagnostics) in 96-well plates, using HOT FIREPol EvaGreen qPCR Mix (Solis BioDyne) in the PCR reaction mixture, according to manufacturer’s instructions (i.e., final volume of 20 μL containing 4 μL of the EvaGreen qPCR mix, 1 μL of 10 μM primers, and 1 μL of a 1/10 dilution of cDNA as template).

    Degradation Assay:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl. .. For the nucleic acid degradation assay, 15 μ g of total RNA, circular plasmid, digested linear plasmid, or genomic DNA prepared from leaves of O. minuta was incubated with the recombinant GST-OmBBD proteins for 2 h at 56°C.

    Construct:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001). ..

    Staining:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl. .. For the nucleic acid degradation assay, 15 μ g of total RNA, circular plasmid, digested linear plasmid, or genomic DNA prepared from leaves of O. minuta was incubated with the recombinant GST-OmBBD proteins for 2 h at 56°C.

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. The nucleus of all cells was stained with Hoechst 33342 (1:100, Sigma-Aldrich) and the nucleus of dead cells were counterstained with propidium iodide (1:100, Invitrogen) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield mounting medium (Vector laboratories).

    In Situ Hybridization:

    Article Title: Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner
    Article Snippet: After treatment with RNase-free DNase I (04716728001, Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C, probes were hydrolyzed in hydrolysis buffer (40 mM NaHCO3 , 60 mM Na2 CO3 ) at 60°C for up to 30 min, depending on probe size. .. Frozen section in situ hybridization: frozen slides were warmed for 20 min at room temperature and then 5 min at 50°C on a slide warmer.

    Plasmid Preparation:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: Vector preps were then buffer exchanged and concentrated into PBS with 0.001% or 0.00025% Tween 20 (Sigma-Aldrich, 9416) using Amicon filtration (EMD Millipore, UFC910024) at 3000 g . .. DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195).

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: The resulting in-frame fusion plasmid was transformed into Escherichia coli BL21 (DE3)-pLys, and expression in E. coli was then induced by the addition of 0.5 m m isopropyl- β - d -thiogalactoside at 30°C for 3 h. The recombinant OmBBD-GST proteins were purified using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and 10 m m reduced glutathione (Sigma) dissolved in 50 m m Tris-HCl (pH 8.0). .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks. .. The nucleus of all cells was stained with Hoechst 33342 (1:100, Sigma-Aldrich) and the nucleus of dead cells were counterstained with propidium iodide (1:100, Invitrogen) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield mounting medium (Vector laboratories).

    In Vitro:

    Article Title: Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner
    Article Snippet: Restriction digest and in vitro transcription were done according to manufacturer’s instructions, with DIG RNA Labeling Mix (11277073910, Sigma-Aldrich, St. Louis, MO). .. After treatment with RNase-free DNase I (04716728001, Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C, probes were hydrolyzed in hydrolysis buffer (40 mM NaHCO3 , 60 mM Na2 CO3 ) at 60°C for up to 30 min, depending on probe size.

    Real-time Polymerase Chain Reaction:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: .. DNase-resistant (Sigma-Aldrich, 04536282001) viral genomic titers were measured by real-time qPCR( ) using SYBR Green I (Invitrogen, S7567) and the CFX96 Real-Time PCR cycler (Bio-Rad, 1855195). .. To synthesize sgRNAs in vitro , a DNA template that contained a T7 promoter, a 20-nt guide sequence, and a sgRNA scaffold was generated by overlapping PCR.

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). .. RT-qPCR was performed with a LightCycler 96 Real-Time PCR Systtem (Roche Diagnostics) in 96-well plates, using HOT FIREPol EvaGreen qPCR Mix (Solis BioDyne) in the PCR reaction mixture, according to manufacturer’s instructions (i.e., final volume of 20 μL containing 4 μL of the EvaGreen qPCR mix, 1 μL of 10 μM primers, and 1 μL of a 1/10 dilution of cDNA as template).

    Negative Control:

    Article Title: Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model
    Article Snippet: A negative control in which the TdT was omitted was included in each trial. .. The positive control was performed incubating the cells with DNase I recombinant (Sigma-Aldrich) for 10 minutes before to labeling procedures to induce DNA stands breaks.

    RNA Expression:

    Article Title: Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease
    Article Snippet: Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I (rDNase I; Sigma). .. A stem-loop reverse transcription-quantitative PCR (RT-qPCR) method was used to quantitate miR-like RNA expression as in a previous study ( ).

    Agarose Gel Electrophoresis:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl. .. Degraded nucleic acids were electrophoresed on a 1.0% agarose gel for RNA or a 0.8% gel for DNA and then stained with ethidium bromide ( ).

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN), and RNA quantity and integrity were assessed using both a Nanodrop spectrophotometer (Thermo Scientific), and standard agarose-gel electrophoretic analysis. .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen).

    In Situ:

    Article Title: Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner
    Article Snippet: RNA in situ hybridization Probe preparation: mouse cDNA plasmids containing the following inserts were used to make RNA in situ probes, and were cut and transcribed with the indicated restriction enzyme (New England Biolabs, Ipswich, MA) and RNA polymerase (New England Biolabs, Ipswich, MA): Fgfr1 transmembrane domain (pBluescriptKS-Fgfr1TM , 325 bp, HincII, T7, gift of K. Peters), Fgf20 (pGEMT-Fgf20 , 653 bp, NcoI, Sp6), Sox2 (pBluescriptSK-Sox2 , 750 bp, AccI, T3, gift of A. Kiernan), Etv4 (pGEM-Etv4 , ~2300 bp, ApaI, Sp6, gift of G. Martin), Etv5 (pBluescriptSK-Etv5 , ~4000 bp, HindIII, T3, gift of G. Martin), Hey1 (pT7T3D-Hey1 [IMAGE clone #478014], 343 bp, EcoRI, T3, gift of S. Rentschler), Hey2 (pCMVSPORT6-Hey2 [IMAGE clone #5374813], 819 bp, EcoRI, T7, gift of S. Rentschler). .. After treatment with RNase-free DNase I (04716728001, Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C, probes were hydrolyzed in hydrolysis buffer (40 mM NaHCO3 , 60 mM Na2 CO3 ) at 60°C for up to 30 min, depending on probe size.

    Electrophoresis:

    Article Title: Novel Bifunctional Nucleases, OmBBD and AtBBD1, Are Involved in Abscisic Acid-Mediated Callose Deposition in Arabidopsis
    Article Snippet: After electrophoresis, the gel was washed with 25% isopropanol in 0.01 m Tris-HCl. .. For the detection of DNase activity, the gel was incubated in 0.1 m Tris-HCl buffer containing 50 μ m MnCl2 , 50 μ m CaCl2 , 50 μ m ZnCl2 , and 50 μ m MgCl2 at 45°C for 3 h. The gels were then stained with 0.2% toluidine blue O in 0.01 m Tris-HCl (Sigma) for 20 min and destained in 0.01 m Tris-HCl.

    Spectrophotometry:

    Article Title: The Arabidopsis bZIP19 and bZIP23 Activity Requires Zinc Deficiency – Insight on Regulation From Complementation Lines
    Article Snippet: Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN), and RNA quantity and integrity were assessed using both a Nanodrop spectrophotometer (Thermo Scientific), and standard agarose-gel electrophoretic analysis. .. The RNA samples were treated with Recombinant DNase I (Sigma-Aldrich) and first strand cDNA synthesis was generated from 0.5 μg total RNA using a SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen).

    Evaporation:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: A piece of glass slide or parafilm was used to cover the top facet of the flow cell, preventing the imaging or hybridization buffer from evaporation. .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes.

    Concentration Assay:

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: Cells or tissue sections within flow chambers were hybridized at a concentration of 1nM per probe overnight in a hybridization buffer of 10% Dextran Sulfate (Sigma D8906), 30% Formamide, 2X Saline-Sodium Citrate (SSC) buffer at room temperature. .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: When the OD at 600 nm reached 0.6, 1 M IPTG was added to a final concentration of 0.5 mM. .. To lyse the bacteria, cell pellets were suspended in 5 mL (for Sph1, Sph3, and Sph4) or 10 mL (Sph2) of BugBuster (EMD Biosciences) containing 20 unit/mL DNase I (Thermo Scientific) and 0.25 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and the suspension was swirled for 20 min at room temperature.

    Lysis:

    Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
    Article Snippet: .. For all TPX2 constructs, cells were lysed using an EmulsiFlex (Avestin) in lysis buffer (0.05 M Tris-HCl, 0.015 M Imidazole, 0.75 M NaCl, pH 7.75) containing 0.0002 M phenylmethylsulfonyl fluoride (PMSF), 0.006 M β-mercaptoethanol (βME), cOmplete™ EDTA-free Protease Inhibitor tablet (Sigma 5056489001), and 1000 U DNase I (Sigma 04716728001). ..

    Fluorescence In Situ Hybridization:

    Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells
    Article Snippet: .. Probes were labeled for use in FISH by nick translation as follows: prior to nick translation, 1 μg DNA was treated with RNase (0.02 U) (Sigma), for 30 min at 37°C, nick translation was carried out at 16°C for 1 h in the following reaction mixture; 50 mM Tris-HCl, 5 mM MgCl2 , 2.5 μg BSA, 10 mM β-mercaptoethanol, 50 mM dAGC, 20 μM hapten/fluor [digoxigenin-11-dUTP (Sigma); Cy3 dUTP (GE Healthcare)], 15 U recombinant DNase1 (Sigma) and 10 U DNA polymerase I (NEB), made up to a final volume of 50 μL with H2 0. .. Imaging Equipment and Settings Widefield fluorescence imaging was performed at 20°C on a DeltaVision Elite system (Applied Precision) equipped with a 100x/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40), FITC (excitation 475/28; emission 525/45) and TRITC (excitation 542/27; emission 593/45) filters.

    Article Title: Dense transcript profiling in single cells by image correlation decoding
    Article Snippet: .. After imaging the samples, FISH probes were removed by 100 Units of DNase I enzymatic treatment (Sigma-Aldrich, 04716728001 ROCHE) for 4 hours followed by a post wash with 30% formamide and 2X SSC wash for 10 minutes. .. The cells were subsequently hybridized by another probe set at 1 nM concentration for more than 12 hours at room temperate in the hybridization buffer.

    Affinity Column:

    Article Title: Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships
    Article Snippet: Pellets were lysed with 1x Bugbuster (Novagen) and DNase for 30 min at room temperature and then centrifuged at 4°C under high speed to collect the supernatant. .. The supernatant was loaded onto a nickel affinity column and was washed with 20 mM Tris, 1 mM DTT, 300 mM KCl (pH 8.0).

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