i rfi dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher i rfi dna
    MoMLV PIC is compacted by BAF. (A) Sedimentation properties of initial (panel 1), salt-stripped (panel 2), BAF-reconstituted (panel 3), HMGA1-reconstituted (panels 4 and 5), and HMGB1-reconstituted (panels 6 and 7) PICs. The MoMLV PIC fraction was salt stripped with 750 mM KCl and then incubated with BAF, HMGA1, or HMGB1 protein in the presence of 400 mM KCl (panels 1, 2, 3, 4, and 6) or 150 mM KCl (panels 5 and 7). After gel filtration, each reconstituted PIC fraction was layered on a 15 to 30% sucrose gradient and centrifuged at 30,000 rpm for 1 h with a Beckman rotor. The gradient was fractionated from the top to bottom into 22 fractions, and viral <t>DNA</t> in each fraction was detected by Southern blotting. (B) Intermolecular integration activities of initial, salt-stripped, and reconstituted PICs. Salt-stripped PICs were incubated with BAF (lane 3), HMGA1 (lanes 4 and 6), or HMGB1 (lanes 5 and 7) in the presence of 400 mM KCl (lanes 1 to 5) or 150 mM KCl (lanes 6 and 7) and then added to the integration reaction mixture containing φX174 <t>RFI</t> DNA as the target DNA to check integration activities. After incubation, DNA products from the reaction were digested with Bam HI and detected by Southern blotting with a 32 ).
    I Rfi Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    φx174 replicative

    Images

    1) Product Images from "Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes"

    Article Title: Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.23.12376-12380.2002

    MoMLV PIC is compacted by BAF. (A) Sedimentation properties of initial (panel 1), salt-stripped (panel 2), BAF-reconstituted (panel 3), HMGA1-reconstituted (panels 4 and 5), and HMGB1-reconstituted (panels 6 and 7) PICs. The MoMLV PIC fraction was salt stripped with 750 mM KCl and then incubated with BAF, HMGA1, or HMGB1 protein in the presence of 400 mM KCl (panels 1, 2, 3, 4, and 6) or 150 mM KCl (panels 5 and 7). After gel filtration, each reconstituted PIC fraction was layered on a 15 to 30% sucrose gradient and centrifuged at 30,000 rpm for 1 h with a Beckman rotor. The gradient was fractionated from the top to bottom into 22 fractions, and viral DNA in each fraction was detected by Southern blotting. (B) Intermolecular integration activities of initial, salt-stripped, and reconstituted PICs. Salt-stripped PICs were incubated with BAF (lane 3), HMGA1 (lanes 4 and 6), or HMGB1 (lanes 5 and 7) in the presence of 400 mM KCl (lanes 1 to 5) or 150 mM KCl (lanes 6 and 7) and then added to the integration reaction mixture containing φX174 RFI DNA as the target DNA to check integration activities. After incubation, DNA products from the reaction were digested with Bam HI and detected by Southern blotting with a 32 ).
    Figure Legend Snippet: MoMLV PIC is compacted by BAF. (A) Sedimentation properties of initial (panel 1), salt-stripped (panel 2), BAF-reconstituted (panel 3), HMGA1-reconstituted (panels 4 and 5), and HMGB1-reconstituted (panels 6 and 7) PICs. The MoMLV PIC fraction was salt stripped with 750 mM KCl and then incubated with BAF, HMGA1, or HMGB1 protein in the presence of 400 mM KCl (panels 1, 2, 3, 4, and 6) or 150 mM KCl (panels 5 and 7). After gel filtration, each reconstituted PIC fraction was layered on a 15 to 30% sucrose gradient and centrifuged at 30,000 rpm for 1 h with a Beckman rotor. The gradient was fractionated from the top to bottom into 22 fractions, and viral DNA in each fraction was detected by Southern blotting. (B) Intermolecular integration activities of initial, salt-stripped, and reconstituted PICs. Salt-stripped PICs were incubated with BAF (lane 3), HMGA1 (lanes 4 and 6), or HMGB1 (lanes 5 and 7) in the presence of 400 mM KCl (lanes 1 to 5) or 150 mM KCl (lanes 6 and 7) and then added to the integration reaction mixture containing φX174 RFI DNA as the target DNA to check integration activities. After incubation, DNA products from the reaction were digested with Bam HI and detected by Southern blotting with a 32 ).

    Techniques Used: Sedimentation, Incubation, Filtration, Southern Blot

    Related Articles

    Activity Assay:

    Article Title: Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes
    Article Snippet: .. Integration activities of reconstituted PICs were evaluated by the previously described integration activity assay with φX174 replicative form I (RFI) DNA (Gibco BRL) as the target DNA ( ). .. For the sedimentation assay, PIC fractions were gel filtrated with a spin column equilibrated with buffer B containing 150 mM KCl to remove the Nycodenz from the solution, loaded on 15 to 30% sucrose gradients in buffer A containing 6 mM EDTA, and centrifuged in a Beckman TLS55 rotor at 30,000 rpm for 1 h at 4°C.

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    Thermo Fisher polyacrylamide tbe gels
    ET SSB reduces off-target activity with Tt Ago. Sets of two guides targeting different locations on ΦX174 Virion DNA over increasing concentration of ET SSB. Reactions were carried out at 74 °C for 5 hr and products were analyzed on <t>Novex</t> 20% polyacrylamide <t>TBE</t> gels stained with 3X GelRed in water. Off target cutting is reduced with higher concentrations of ET SSB. ( A ) 528 nt product using ΦX174 guides 3 and 5; ( B ) 728 nt product using ΦX174 guides 3 and 6; ( C ) 359 nt product using ΦX174 guides 1 and 4; ( D ) 779 nt product using ΦX174 guides 2 and 6.
    Polyacrylamide Tbe Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ET SSB reduces off-target activity with Tt Ago. Sets of two guides targeting different locations on ΦX174 Virion DNA over increasing concentration of ET SSB. Reactions were carried out at 74 °C for 5 hr and products were analyzed on Novex 20% polyacrylamide TBE gels stained with 3X GelRed in water. Off target cutting is reduced with higher concentrations of ET SSB. ( A ) 528 nt product using ΦX174 guides 3 and 5; ( B ) 728 nt product using ΦX174 guides 3 and 6; ( C ) 359 nt product using ΦX174 guides 1 and 4; ( D ) 779 nt product using ΦX174 guides 2 and 6.

    Journal: PLoS ONE

    Article Title: Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro

    doi: 10.1371/journal.pone.0203073

    Figure Lengend Snippet: ET SSB reduces off-target activity with Tt Ago. Sets of two guides targeting different locations on ΦX174 Virion DNA over increasing concentration of ET SSB. Reactions were carried out at 74 °C for 5 hr and products were analyzed on Novex 20% polyacrylamide TBE gels stained with 3X GelRed in water. Off target cutting is reduced with higher concentrations of ET SSB. ( A ) 528 nt product using ΦX174 guides 3 and 5; ( B ) 728 nt product using ΦX174 guides 3 and 6; ( C ) 359 nt product using ΦX174 guides 1 and 4; ( D ) 779 nt product using ΦX174 guides 2 and 6.

    Article Snippet: Novex™ 20% polyacrylamide TBE Gels were obtained from Thermo Fisher Scientific, Inc. (Waltham, Massachusetts, USA).

    Techniques: Activity Assay, Concentration Assay, Staining