φx174  (New England Biolabs)


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    Structured Review

    New England Biolabs φx174
    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 <t>φX174</t> 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.
    φx174, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 "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

    3) 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

    Related Articles

    other:

    Article Title: DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51
    Article Snippet: φX174 form I DNA was from New England BioLabs.

    Plasmid Preparation:

    Article Title: CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides
    Article Snippet: .. LbCas12a protein, circular single-stranded phage DNA M13mp18 (7,249 bases in length), ΦX174 (5,386 bases in length), and circular double-stranded plasmid DNA pUC19 (2,686 bp in length) are obtained from New England Biolabs. .. FnCas12a protein is from Applied Biological Materials.

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    New England Biolabs φx174 ssdna
    DNA synthesis of DNA pol-prim on natural <t>ssDNA</t> in the presence of RPA and PyV Tag. The cooperation of PyV Tag with mouse, human, and mutant pol-prims was tested on natural ssDNA bound by human RPA. In the reaction, <t>ΦX174</t> ssDNA, RPA, and pol-prim complexes as indicated were incubated in the absence (shaded bars) or in the presence (solid bars) of PyV Tag. The incorporation of acid-insoluble dNMPs in the absence of Tag was arbitrarily set to 1. The presented data are the mean values and standard deviations of three experiments.
    φx174 Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs φx174 am3cs70 single stranded virion dna
    <t>DNA</t> sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic <t>φX174</t> molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared
    φx174 Am3cs70 Single Stranded Virion Dna, supplied by New England Biolabs, 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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    88
    New England Biolabs bacteriophage φx174
    High-resolution velocity sedimentation gradients reveal two HCV capsid subpopulations. Higher-resolution 10 to 50% linear sucrose gradients were used to separate wheat germ cell-free reactions programmed with C173, C115, and HBV core transcript. Fractions were analyzed by SDS-PAGE and autoradiography. The graph shows the amount of core in each fraction (quantitated by densitometry) for C173 (solid diamonds), C115 (solid squares), and HBV core (open circles). Arrows indicate the migration of sedimentation markers (80S and 114S); 80S ribosomes were identified by Coomassie staining, and 114S was identified by titering bacteriophage <t>φX174</t> (open triangles). Shown are representative results from three independent experiments.
    Bacteriophage φx174, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA synthesis of DNA pol-prim on natural ssDNA in the presence of RPA and PyV Tag. The cooperation of PyV Tag with mouse, human, and mutant pol-prims was tested on natural ssDNA bound by human RPA. In the reaction, ΦX174 ssDNA, RPA, and pol-prim complexes as indicated were incubated in the absence (shaded bars) or in the presence (solid bars) of PyV Tag. The incorporation of acid-insoluble dNMPs in the absence of Tag was arbitrarily set to 1. The presented data are the mean values and standard deviations of three experiments.

    Journal: Journal of Virology

    Article Title: Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase ?-Primase To Synthesize DNA In Vitro

    doi: 10.1128/JVI.75.18.8569-8578.2001

    Figure Lengend Snippet: DNA synthesis of DNA pol-prim on natural ssDNA in the presence of RPA and PyV Tag. The cooperation of PyV Tag with mouse, human, and mutant pol-prims was tested on natural ssDNA bound by human RPA. In the reaction, ΦX174 ssDNA, RPA, and pol-prim complexes as indicated were incubated in the absence (shaded bars) or in the presence (solid bars) of PyV Tag. The incorporation of acid-insoluble dNMPs in the absence of Tag was arbitrarily set to 1. The presented data are the mean values and standard deviations of three experiments.

    Article Snippet: The DNA replication of ΦX174 ssDNA was carried out in a reaction mixture containing 66 ng of ΦX174 ssDNA (New England Biolabs); 20 mM Tris-acetate (pH 7.3); 5 mM magnesium acetate; 20 mM potassium acetate; 1 mM dithiothreitol; 0.1 mg of bovine serum albumin (BSA)/ml; 1 mM ATP; 0.1 mM (each) CTP, GTP, UTP, dATP, dCTP, dGTP, and TTP; and 0.1 mM [α-32 P]dCTP (Amersham Pharmacia Biotech) ( ).

    Techniques: DNA Synthesis, Recombinase Polymerase Amplification, Mutagenesis, Incubation

    Ca 2+ stimulates DNA three-strand exchange promoted by hRad51 protein. ( a ) The scheme and the time course of DNA stand exchange between φX174 circular ssDNA and linear dsDNA promoted by hRad51 protein analyzed in a 1% agarose gel. The reactions

    Journal:

    Article Title: Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

    doi: 10.1073/pnas.0402105101

    Figure Lengend Snippet: Ca 2+ stimulates DNA three-strand exchange promoted by hRad51 protein. ( a ) The scheme and the time course of DNA stand exchange between φX174 circular ssDNA and linear dsDNA promoted by hRad51 protein analyzed in a 1% agarose gel. The reactions

    Article Snippet: Oligonucleotides, pUC19 DNA, and εDNA were prepared as described ( , ). φX174 ssDNA and dsDNA were purchased from New England Biolabs and Invitrogen, respectively.

    Techniques: Agarose Gel Electrophoresis

    DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

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

    Article Title: Cell-free cloning using ?29 DNA polymerase

    doi: 10.1073/pnas.0508809102

    Figure Lengend Snippet: DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

    Article Snippet: DNA Preparations. φX174 am3cs70 single-stranded virion DNA and M13mp18 single-stranded virion DNA were obtained from NEB (Beverly, MA).

    Techniques: DNA Sequencing, Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification

    High-resolution velocity sedimentation gradients reveal two HCV capsid subpopulations. Higher-resolution 10 to 50% linear sucrose gradients were used to separate wheat germ cell-free reactions programmed with C173, C115, and HBV core transcript. Fractions were analyzed by SDS-PAGE and autoradiography. The graph shows the amount of core in each fraction (quantitated by densitometry) for C173 (solid diamonds), C115 (solid squares), and HBV core (open circles). Arrows indicate the migration of sedimentation markers (80S and 114S); 80S ribosomes were identified by Coomassie staining, and 114S was identified by titering bacteriophage φX174 (open triangles). Shown are representative results from three independent experiments.

    Journal: Journal of Virology

    Article Title: Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

    doi: 10.1128/JVI.78.17.9257-9269.2004

    Figure Lengend Snippet: High-resolution velocity sedimentation gradients reveal two HCV capsid subpopulations. Higher-resolution 10 to 50% linear sucrose gradients were used to separate wheat germ cell-free reactions programmed with C173, C115, and HBV core transcript. Fractions were analyzed by SDS-PAGE and autoradiography. The graph shows the amount of core in each fraction (quantitated by densitometry) for C173 (solid diamonds), C115 (solid squares), and HBV core (open circles). Arrows indicate the migration of sedimentation markers (80S and 114S); 80S ribosomes were identified by Coomassie staining, and 114S was identified by titering bacteriophage φX174 (open triangles). Shown are representative results from three independent experiments.

    Article Snippet: Bacteriophage φX174 and E. coli strain H4714 were gifts from P. Gouldfarb, New England Biolabs.

    Techniques: Sedimentation, SDS Page, Autoradiography, Migration, Staining