φx174  (New England Biolabs)


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    Name:
    DNase I RNase free
    Description:
    DNase I RNase free 5 000 units
    Catalog Number:
    M0303L
    Price:
    282
    Category:
    Deoxyribonucleases DNase
    Size:
    5 000 units
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    Structured Review

    New England Biolabs φx174
    DNase I RNase free
    DNase I RNase free 5 000 units
    https://www.bioz.com/result/φx174/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    φx174 - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51"

    Article Title: DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh853

    Rad22-promoted D-loop formation and D-loop cleavage by Mus81. ( A ) Schematic of D-loop formation. The asterisk indicates the position of the 5′- 32 P-end-label on the partial duplex. ( B ) Rad22-promoted D-loop formation. Reactions are described in Materials and Methods and contained Rad22 (6 nM, lanes b and e; 12 nM, lanes c and f; and 24 nM, lanes d and g) and 10 mM MgCl 2 . Rad22 was pre-incubated with partial duplex or φX174 DNA as indicated. ( C ) Dependence on homology for D-loop formation. Reactions are described in Materials and Methods. ( D ) Effect of Mus81 on Rad22-promoted φX174-based D-loop formation. Reactions contained 12 nM Rad22 and 14 nM Mus81–Eme1 as indicated. ( E ) Cleavage of purified φX174-based D-loops by Mus81–Eme1. Reactions contained 7 nM (lane c) or 14 nM (lanes d and e) Mus81–Eme1 and 10 mM MgCl 2 as indicated. The D-loop (lane a) was dissociated by heat treatment at 96°C for 2 min.
    Figure Legend Snippet: Rad22-promoted D-loop formation and D-loop cleavage by Mus81. ( A ) Schematic of D-loop formation. The asterisk indicates the position of the 5′- 32 P-end-label on the partial duplex. ( B ) Rad22-promoted D-loop formation. Reactions are described in Materials and Methods and contained Rad22 (6 nM, lanes b and e; 12 nM, lanes c and f; and 24 nM, lanes d and g) and 10 mM MgCl 2 . Rad22 was pre-incubated with partial duplex or φX174 DNA as indicated. ( C ) Dependence on homology for D-loop formation. Reactions are described in Materials and Methods. ( D ) Effect of Mus81 on Rad22-promoted φX174-based D-loop formation. Reactions contained 12 nM Rad22 and 14 nM Mus81–Eme1 as indicated. ( E ) Cleavage of purified φX174-based D-loops by Mus81–Eme1. Reactions contained 7 nM (lane c) or 14 nM (lanes d and e) Mus81–Eme1 and 10 mM MgCl 2 as indicated. The D-loop (lane a) was dissociated by heat treatment at 96°C for 2 min.

    Techniques Used: Incubation, Purification

    2) Product Images from "CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides"

    Article Title: CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.12.038

    Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.
    Figure Legend Snippet: Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.

    Techniques Used: Activity Assay, Concentration Assay

    3) Product Images from "Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast"

    Article Title: Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh720

    Pfh1 helicase unwinds short duplex DNA in a distributive manner. ( A ) The oligonucleotides indicated were annealed to ΦX174 sscDNA to construct the 30, 40 or 52 bp partial duplex substrates as shown. The reaction mixtures (200 μl) were
    Figure Legend Snippet: Pfh1 helicase unwinds short duplex DNA in a distributive manner. ( A ) The oligonucleotides indicated were annealed to ΦX174 sscDNA to construct the 30, 40 or 52 bp partial duplex substrates as shown. The reaction mixtures (200 μl) were

    Techniques Used: Construct

    Related Articles

    Footprinting:

    Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding
    Article Snippet: After incubation at 22°C for 10 min, the binding reaction mixtures were loaded on 5% (w/v) native polyacrylamide gels and run in TBE buffer at 100 V for 45 min. EMSA data were collected and analysed on a PhosphoImager (FujiFilm) using Multi Gauge image analysis software (FujiFilm). .. DNase I footprinting Templates for DNase I footprinting were amplified by PCR using one unlabelled primer and one primer 5′-end labelled using [γ32 -P] ATP and T4 polynucleotide kinase (New England Biolabs). ..

    Amplification:

    Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding
    Article Snippet: After incubation at 22°C for 10 min, the binding reaction mixtures were loaded on 5% (w/v) native polyacrylamide gels and run in TBE buffer at 100 V for 45 min. EMSA data were collected and analysed on a PhosphoImager (FujiFilm) using Multi Gauge image analysis software (FujiFilm). .. DNase I footprinting Templates for DNase I footprinting were amplified by PCR using one unlabelled primer and one primer 5′-end labelled using [γ32 -P] ATP and T4 polynucleotide kinase (New England Biolabs). ..

    Polymerase Chain Reaction:

    Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding
    Article Snippet: After incubation at 22°C for 10 min, the binding reaction mixtures were loaded on 5% (w/v) native polyacrylamide gels and run in TBE buffer at 100 V for 45 min. EMSA data were collected and analysed on a PhosphoImager (FujiFilm) using Multi Gauge image analysis software (FujiFilm). .. DNase I footprinting Templates for DNase I footprinting were amplified by PCR using one unlabelled primer and one primer 5′-end labelled using [γ32 -P] ATP and T4 polynucleotide kinase (New England Biolabs). ..

    Lysis:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: Identification of ligated RNAs This study (Fig. ) employed in vivo crosslinking of RNA duplexes with the AMT molecule, which can, upon 365 nm UV irradiation, generate inter-strand adducts between juxtaposed uridine bases [ ]. .. Following cell lysis and RNA extraction, DNA residues were digested by DNase I, and single strand RNAs and free RNA overhangs adjacent to duplexes were digested by RNase T1. .. Then, the residual single strand RNAs were hybridized with 20 nt oligonucleotides and digested by RNase H three times.

    RNA Extraction:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: Identification of ligated RNAs This study (Fig. ) employed in vivo crosslinking of RNA duplexes with the AMT molecule, which can, upon 365 nm UV irradiation, generate inter-strand adducts between juxtaposed uridine bases [ ]. .. Following cell lysis and RNA extraction, DNA residues were digested by DNase I, and single strand RNAs and free RNA overhangs adjacent to duplexes were digested by RNase T1. .. Then, the residual single strand RNAs were hybridized with 20 nt oligonucleotides and digested by RNase H three times.

    Binding Assay:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Individual footprint reactions were initiated with a 22 μl binding reaction mixture that contained 12.5 mM HEPES (pH 7.8), 5 mM K-acetate (pH 8.0), 2.5 mM Mg-acetate, 1 mM CaCl2 , 12.5 μg/ml bovine serum albumin [ ], 0.3 mg/ml heparin, 200 nM of fluorescence-labeled DNA probe and various amounts of purified protein. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion. ..

    Incubation:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Individual footprint reactions were initiated with a 22 μl binding reaction mixture that contained 12.5 mM HEPES (pH 7.8), 5 mM K-acetate (pH 8.0), 2.5 mM Mg-acetate, 1 mM CaCl2 , 12.5 μg/ml bovine serum albumin [ ], 0.3 mg/ml heparin, 200 nM of fluorescence-labeled DNA probe and various amounts of purified protein. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion. ..

    Article Title: Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription
    Article Snippet: .. Nuclei were incubated with various concentrations of Dnase I (0–1.5 units, NEB) for 10 minutes at 37°C. .. Reactions were stopped by adding an equal volume of 2× Lysis Buffer (1% SDS, 200 mM NaCl, 10 mM EDTA, 20 mM Tris pH 7.5, 0.4 mg/ml proteinase K) and incubated overnight at 37°C.

    Synthesized:

    Article Title: Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae
    Article Snippet: The quantity of 5′-methylguanosine-capped RNA was measured by NanoDrop (Thermo Fisher) analysis and its integrity was determined with an Agilent 2100 Bioanalyzer. .. 3′-RACE analysis of CPA-sRNAs using 5′-capped RNA 5′-methylguanosine-capped RNA was treated with DNase I (NEB) to remove any contaminating genomic DNA. cDNA was synthesized in 20 µl reactions by adding the following reagents: 1 µg of 5′-methylguanosine-capped RNA, 50 picomole of 3′-oligo(dT) 20 VN primer, 5 mM of dNTPs, 1 U of RNaseOut (Invitrogen) and 5 U of Superscript III (Invitrogen). ..

    other:

    Article Title: Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿
    Article Snippet: However, treatment of the basolateral conditioned supernatant with DNase I did not significantly change its ability to induce IL-8 secretion (Fig. ).

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  • 95
    New England Biolabs circular single stranded φx174 virion dna
    ComM exhibits 3-stranded branch migration activity on long <t>DNA</t> substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded <t>φX174</t> (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.
    Circular Single Stranded φx174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/circular single stranded φx174 virion dna/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    99
    New England Biolabs circular dsdna
    DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of <t>ssDNA</t> or <t>dsDNA.</t> (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .
    Circular Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs φx174 dna
    Analysis of DNA conformation of rAAV vectors. (A) DNA extracted from the rAAV vectors pAAV-hSyn-EGFP (AAV-50465) and pAAV-CAG-GFP (AAV-37825) were left untreated (Ctrl) or incubated in the presence (37°C + Sac II) or absence (37°C) of the restriction enzyme Sac II and products separated by agarose gel electrophoresis. <t>ΦX174</t> DNA was incubated in the presence of Sac II, Hae III or left untreated. The table lists the size of the viral genomes and the expected products after digestion. (B) Undigested and Sac II digested DNA from plasmid DNA or extracted vDNA was amplified with primers targeting the region surrounding the Sac II cleavage site or gfp as a control. Sac II amplification was normalized to gfp amplification, and fold change of undigested to Sac II digested samples was calculated and plotted. gfp , green fluorescent protein gene; rAAV, recombinant adeno-associated virus; vDNA, virus DNA. Color images are available online.
    φx174 Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/φx174 dna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    φx174 dna - by Bioz Stars, 2021-06
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    99
    New England Biolabs circular φx174 ssdna
    DNA strand exchange activities of D149N and R150Q variants compared to WT RAD51. DNA strand exchange assays were carried out as described in ‘Materials and Methods’ section, using normal salt conditions (100 mM KCl). ( A ) Reaction schematic. Homologous <t>φX174</t> circular <t>ssDNA</t> (SS) and linear dsDNA (LDS) substrates are converted by RAD51 protein into heteroduplex products including joint molecules (JM) and nicked circles (NC), which, following deproteination, are resolved from substrates by electrophoresis on agarose gels. The staging of the reaction involves the preincubation of RAD51 with SS in the presence of ATP, followed by the addition of RPA, followed in turn by the addition of LDS to start the reaction. See ‘Materials and Methods’ section for details. ( B ) Comparison of D149N and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by D149N following reaction initiation by the addition of LDS. ( C ) Quantification of strand exchange products from reactions with D149N and WT, shown in panel (B). ( D ) Comparison of R150Q and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by R150Q following reaction initiation by the addition of LDS. ( E ) Quantification of strand exchange products from reactions with R150Q and WT, shown in panel (D).
    Circular φx174 Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ComM exhibits 3-stranded branch migration activity on long DNA substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded φX174 (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.

    Journal: Nucleic Acids Research

    Article Title: ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

    doi: 10.1093/nar/gky343

    Figure Lengend Snippet: ComM exhibits 3-stranded branch migration activity on long DNA substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded φX174 (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.

    Article Snippet: The long three-strand recombination intermediates were generated by RecA-mediated strand exchange between circular single-stranded φX174 virion DNA (NEB) and PstI-linearized double-stranded φX174 DNA (NEB) essentially as previously described ( ).

    Techniques: Migration, Activity Assay, In Vitro, Incubation

    DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of ssDNA or dsDNA. (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .

    Journal: Molecular Cell

    Article Title: Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

    doi: 10.1016/j.molcel.2016.09.031

    Figure Lengend Snippet: DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of ssDNA or dsDNA. (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .

    Article Snippet: Either circular ssDNA (ΦX174 virion, New England Biolabs) or circular dsDNA (ΦX174 RF I, New England Biolabs) was used for activation.

    Techniques: Binding Assay, Mass Spectrometry, Labeling

    Analysis of DNA conformation of rAAV vectors. (A) DNA extracted from the rAAV vectors pAAV-hSyn-EGFP (AAV-50465) and pAAV-CAG-GFP (AAV-37825) were left untreated (Ctrl) or incubated in the presence (37°C + Sac II) or absence (37°C) of the restriction enzyme Sac II and products separated by agarose gel electrophoresis. ΦX174 DNA was incubated in the presence of Sac II, Hae III or left untreated. The table lists the size of the viral genomes and the expected products after digestion. (B) Undigested and Sac II digested DNA from plasmid DNA or extracted vDNA was amplified with primers targeting the region surrounding the Sac II cleavage site or gfp as a control. Sac II amplification was normalized to gfp amplification, and fold change of undigested to Sac II digested samples was calculated and plotted. gfp , green fluorescent protein gene; rAAV, recombinant adeno-associated virus; vDNA, virus DNA. Color images are available online.

    Journal: Human Gene Therapy

    Article Title: A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

    doi: 10.1089/hum.2019.277

    Figure Lengend Snippet: Analysis of DNA conformation of rAAV vectors. (A) DNA extracted from the rAAV vectors pAAV-hSyn-EGFP (AAV-50465) and pAAV-CAG-GFP (AAV-37825) were left untreated (Ctrl) or incubated in the presence (37°C + Sac II) or absence (37°C) of the restriction enzyme Sac II and products separated by agarose gel electrophoresis. ΦX174 DNA was incubated in the presence of Sac II, Hae III or left untreated. The table lists the size of the viral genomes and the expected products after digestion. (B) Undigested and Sac II digested DNA from plasmid DNA or extracted vDNA was amplified with primers targeting the region surrounding the Sac II cleavage site or gfp as a control. Sac II amplification was normalized to gfp amplification, and fold change of undigested to Sac II digested samples was calculated and plotted. gfp , green fluorescent protein gene; rAAV, recombinant adeno-associated virus; vDNA, virus DNA. Color images are available online.

    Article Snippet: DNA concentration and purity were determined by using the Nanodrop spectrophotometer, and the samples were stored at −20°C. rAAV DNA extracts or ΦX174 DNA (New England Biolabs) were digested with Sac II (New England Biolabs) and HaeIII (New England Biolabs) according to the manufacturer's instructions.

    Techniques: Incubation, Agarose Gel Electrophoresis, Plasmid Preparation, Amplification, Recombinant

    DNA strand exchange activities of D149N and R150Q variants compared to WT RAD51. DNA strand exchange assays were carried out as described in ‘Materials and Methods’ section, using normal salt conditions (100 mM KCl). ( A ) Reaction schematic. Homologous φX174 circular ssDNA (SS) and linear dsDNA (LDS) substrates are converted by RAD51 protein into heteroduplex products including joint molecules (JM) and nicked circles (NC), which, following deproteination, are resolved from substrates by electrophoresis on agarose gels. The staging of the reaction involves the preincubation of RAD51 with SS in the presence of ATP, followed by the addition of RPA, followed in turn by the addition of LDS to start the reaction. See ‘Materials and Methods’ section for details. ( B ) Comparison of D149N and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by D149N following reaction initiation by the addition of LDS. ( C ) Quantification of strand exchange products from reactions with D149N and WT, shown in panel (B). ( D ) Comparison of R150Q and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by R150Q following reaction initiation by the addition of LDS. ( E ) Quantification of strand exchange products from reactions with R150Q and WT, shown in panel (D).

    Journal: Nucleic Acids Research

    Article Title: Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

    doi: 10.1093/nar/gku1337

    Figure Lengend Snippet: DNA strand exchange activities of D149N and R150Q variants compared to WT RAD51. DNA strand exchange assays were carried out as described in ‘Materials and Methods’ section, using normal salt conditions (100 mM KCl). ( A ) Reaction schematic. Homologous φX174 circular ssDNA (SS) and linear dsDNA (LDS) substrates are converted by RAD51 protein into heteroduplex products including joint molecules (JM) and nicked circles (NC), which, following deproteination, are resolved from substrates by electrophoresis on agarose gels. The staging of the reaction involves the preincubation of RAD51 with SS in the presence of ATP, followed by the addition of RPA, followed in turn by the addition of LDS to start the reaction. See ‘Materials and Methods’ section for details. ( B ) Comparison of D149N and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by D149N following reaction initiation by the addition of LDS. ( C ) Quantification of strand exchange products from reactions with D149N and WT, shown in panel (B). ( D ) Comparison of R150Q and WT activities. Lanes 1–3 and 18–20 contain markers for SS, NC and LDS, respectively. Lanes 4–10 show the timecourse (0–40 min) of DNA strand exchange catalyzed by WT following reaction initiation by the addition of LDS. Lanes 11–17 show the timecourse (0–40 min) of DNA strand exchange catalyzed by R150Q following reaction initiation by the addition of LDS. ( E ) Quantification of strand exchange products from reactions with R150Q and WT, shown in panel (D).

    Article Snippet: Circular φX174 ssDNA (5.4 kb) was purchased from New England Biolabs.

    Techniques: Electrophoresis, Recombinase Polymerase Amplification

    Properties of hRAD51 variant nucleoprotein filaments. ( A – D ) Electron micrographs of representative nucleoprotein filaments formed by variant and WT RAD51 proteins on φX174 circular ssDNA in the presence of ATP. All images are 100 000X magnification. The black scale bar in each panel represents a distance of 100 nm. ( E ) Differential electrophoretic mobilities of complexes formed by variant and WT RAD51 proteins on φX174 circular ssDNA and linear dsDNA in the presence of ATP. Lanes 1 and 6 contain markers for naked ssDNA and dsDNA, respectively. Lanes 2–5 contain WT, R150Q, D149N and G151D complexes with ssDNA, respectively. Lanes 7–10 contain WT, R150Q, D149N and G151D complexes with dsDNA, respectively. ‘LM’ and ‘HM’ denote low- and high-mobility protein–ssDNA complexes, respectively.

    Journal: Nucleic Acids Research

    Article Title: Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

    doi: 10.1093/nar/gku1337

    Figure Lengend Snippet: Properties of hRAD51 variant nucleoprotein filaments. ( A – D ) Electron micrographs of representative nucleoprotein filaments formed by variant and WT RAD51 proteins on φX174 circular ssDNA in the presence of ATP. All images are 100 000X magnification. The black scale bar in each panel represents a distance of 100 nm. ( E ) Differential electrophoretic mobilities of complexes formed by variant and WT RAD51 proteins on φX174 circular ssDNA and linear dsDNA in the presence of ATP. Lanes 1 and 6 contain markers for naked ssDNA and dsDNA, respectively. Lanes 2–5 contain WT, R150Q, D149N and G151D complexes with ssDNA, respectively. Lanes 7–10 contain WT, R150Q, D149N and G151D complexes with dsDNA, respectively. ‘LM’ and ‘HM’ denote low- and high-mobility protein–ssDNA complexes, respectively.

    Article Snippet: Circular φX174 ssDNA (5.4 kb) was purchased from New England Biolabs.

    Techniques: Variant Assay