t7 helicase  (New England Biolabs)


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    Name:
    T7 DNA Polymerase unmodified
    Description:
    T7 DNA Polymerase unmodified 1 500 units
    Catalog Number:
    m0274l
    Price:
    281
    Size:
    1 500 units
    Category:
    DNA Polymerases
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    Structured Review

    New England Biolabs t7 helicase
    T7 DNA Polymerase unmodified
    T7 DNA Polymerase unmodified 1 500 units
    https://www.bioz.com/result/t7 helicase/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    t7 helicase - by Bioz Stars, 2020-05
    99/100 stars

    Images

    1) Product Images from "DNA looping mediates nucleosome transfer"

    Article Title: DNA looping mediates nucleosome transfer

    Journal: Nature Communications

    doi: 10.1038/ncomms13337

    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).
    Figure Legend Snippet: Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Techniques Used:

    2) Product Images from "DNA looping mediates nucleosome transfer"

    Article Title: DNA looping mediates nucleosome transfer

    Journal: Nature Communications

    doi: 10.1038/ncomms13337

    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).
    Figure Legend Snippet: Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Techniques Used:

    Related Articles

    In Vitro:

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries
    Article Snippet: .. A pair of mutagenic oligonucleotides are phosphorylated and annealed to the ssDNA template for in vitro synthesis of heteroduplex, double-stranded DNA (dsDNA) with T7 DNA polymerase and T4 DNA ligase. .. The resulting product is purified and evaluated by agarose gel electrophoresis; as seen in , the ssDNA can be quantitatively converted to the larger dsDNA species by this technique.

    Ligation:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

    Mutagenesis:

    Article Title: 3? to 5? Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase Modulates Its Strand Displacement Activity
    Article Snippet: .. As a means for comparison, we followed the strand displacement rates for wild-type T7 DNA polymerase holoenzyme (New England Biolabs, Beverly, Mass.) or an exonuclease-deficient mutant T7 DNA polymerase holoenzyme (Sequenase version 2.0; USB, Inc., Cleveland, Ohio). ..

    Incubation:

    Article Title: DNA looping mediates nucleosome transfer
    Article Snippet: .. Replisomes were formed by pre-incubating 1 unit per μl T7 DNA polymerase (NEB) and 1 μM T7 helicase in reaction buffer on ice for 5 min, and then were added to a final concentration of 0.1 unit per μl T7 DNA polymerase (NEB) and 100 nM T7 helicase and incubated at 37 °C for 10 min. .. Samples were then buffer exchanged into 1 × NEBuffer 1 (NEB) using Amicon Ultra-0.5 centrifugal filter units with Ultracel-30 membranes (Millipore).

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
    Article Snippet: .. The template (2 μl of a 10 μM solution) was then incubated with end-labelled 32 P primer 5′ AATGGGCGGAGAGAG 3′ (2 μl of a 10 μM solution), dNTPs (2 μl each of a 1 mM solution), and with either DNA Polymerase I, T7 DNA Polymerase, or T4 DNA Polymerase (1 μl, five units) in the appropriate buffer (5 μl of a 10× solution provided by New England Biolabs with each enzyme), and the reaction was brought to a final volume of 50 μl with dH2 O (38 μl). ..

    Purification:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

    Concentration Assay:

    Article Title: DNA looping mediates nucleosome transfer
    Article Snippet: .. Replisomes were formed by pre-incubating 1 unit per μl T7 DNA polymerase (NEB) and 1 μM T7 helicase in reaction buffer on ice for 5 min, and then were added to a final concentration of 0.1 unit per μl T7 DNA polymerase (NEB) and 100 nM T7 helicase and incubated at 37 °C for 10 min. .. Samples were then buffer exchanged into 1 × NEBuffer 1 (NEB) using Amicon Ultra-0.5 centrifugal filter units with Ultracel-30 membranes (Millipore).

    Protein Extraction:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

    Expressing:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

    Polymerase Chain Reaction:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

    Gel Extraction:

    Article Title: Protein and Antibody Engineering by Phage Display
    Article Snippet: .. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, catalog #: T0440) 0.5 M H2 SO4 Anti-M13-HRP conjugated antibody (GE Healthcare, Marlborough, MA, catalog #: GE27-9421-01) T7 DNA Polymerase (NEB, Ipswich, MA, catalog #: M0274L) T4 DNA Ligase (Invitrogen, Carlsbad, CA, catalog #: 15224-017) 25 m M dNTPs (Fisher, Hampton, NH, catalog #: FERR1121) 25 mg/mL Uridine (Sigma, St. Louis, MO, catalog #: U3750-100G) BugBuster 10X Protein Extraction Reagent (Millipore, Billerica, MA, catalog #: 70921) QIAquick PCR Purification Kit (Qiagen, Hilden, Germany, catalog #: 28104) QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, catalog #: 28704) QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog #: 27104) NucleoBond Xtra Maxi Plus (Macherey-Nagel, Düren, Germany, catalog #: 740416.50) Ni-NTA Agarose (Qiagen, Hilden, Germany, catalog #: 30210) DNaseI (Invitrogen, Carlsbad, CA, catalog #: 18047-019) SIGMAFast EDTA-free protease cocktail inhibitor (Sigma, St. Louis, MO, catalog #: S8830) Precision Plus Protein Dual Color Standards (BioRad, Hercules, CA, catalog #: 161-03741) Anti-His (C-term)-HRP antibody (Invitrogen, Carlsbad, CA, catalog #: 46-0707) Protein A Agarose (Pierce, Thermo Fisher Scientific, Waltham, MA, catalog #: 15918-014) Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA, catalog #: M0531S) Restriction enzymes: BssHII, BsiWI, NheI, NsiI, FseI (NEB, Ipswich, MA, catalog #s: R0199S, R0553S, R0131S, R0127S, R0558S, respectively) Alkaline Phosphatase, Calf Intestinal (NEB, Ipswich, MA, catalog #: M0290S) Quick Ligation Kit (NEB, Ipswich, MA, catalog #: M2200S) Polyethylenimine (PEI) Linear, 25,000 MW (Polysciences, Warrington, PA, catalog #: 23966) FreeStyle™ 293 Expression Medium (ThermoFisher; Invitrogen, Carlsbad, CA, catalog #: 12338-018) M2 antibody (Sigma-Aldrich, St. Louis, MO, catalog #: F1804) Protein A-HRP conjugate (Invitrogen, Carlsbad, CA, catalog #: 101023) ..

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    New England Biolabs t7 helicase
    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a <t>T7</t> helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).
    T7 Helicase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 helicase/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    t7 helicase - by Bioz Stars, 2020-05
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    92
    New England Biolabs primase assay
    RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) <t>Primase</t> assay. RPA was pre-bound to unprimed (left) and primed (right) M13mp18 ssDNA for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.
    Primase Assay, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs dna polymerase α primase
    Initiation of SV40 <t>DNA</t> replication in vitro by DNA polymerase <t>α-primase</t> (pol-prim) prephosphorylated by cyclin A/cdk2 or cyclin E/cdk2. (A) Increasing amounts of the indicated preparations of DNA polymerase α-primase were tested for initiation of SV40 DNA replication. The initiation products of each reaction were separated by denaturing PAGE and quantitated by PhosphorImager analysis, and the background (data not shown) was subtracted to give the number of SV40 initiation units per microliter of DNA polymerase α-primase. The bar to the right indicates primers of 8 to 10 nucleotides. (B) The SV40 initiation activity in each reaction in A was divided by the number of primase units per microliter (data not shown) determined for the corresponding preparation of phosphorylated DNA polymerase α-primase. The bar graph shows the average relative initiation activity of each preparation; that of the mock-phosphorylated preparation was set to 100%. The error bars represent the variation obtained from three different reactions with each preparation of DNA polymerase α-primase.
    Dna Polymerase α Primase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Journal: Nature Communications

    Article Title: DNA looping mediates nucleosome transfer

    doi: 10.1038/ncomms13337

    Figure Lengend Snippet: Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Article Snippet: Replisomes were formed by pre-incubating 1 unit per μl T7 DNA polymerase (NEB) and 1 μM T7 helicase in reaction buffer on ice for 5 min, and then were added to a final concentration of 0.1 unit per μl T7 DNA polymerase (NEB) and 100 nM T7 helicase and incubated at 37 °C for 10 min.

    Techniques:

    RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) Primase assay. RPA was pre-bound to unprimed (left) and primed (right) M13mp18 ssDNA for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.

    Journal: Molecular Cell

    Article Title: The Initial Response of a Eukaryotic Replisome to DNA Damage

    doi: 10.1016/j.molcel.2018.04.022

    Figure Lengend Snippet: RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) Primase assay. RPA was pre-bound to unprimed (left) and primed (right) M13mp18 ssDNA for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.

    Article Snippet: Primase assay Primed template was prepared by annealing oligonucleotide JY180 (500 nM) (sequence: ) to M13mp18 ssDNA (50 nM) (New England Biolabs) in 10 mM Tris-Cl pH 7.6, 5 mM EDTA and 100 mM NaCl.

    Techniques: Recombinase Polymerase Amplification, Binding Assay, Titration

    Architecture of the DNA–primase complex. ( A ) Crystal structure of the catalytic domain of RepB′ in complex with ssiA (3′Δ13). Catalytic domain in ribbon representation is green, and ssiA (3′Δ13) is shown as

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

    Article Title: Structure and function of primase RepB? encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

    doi: 10.1073/pnas.0902910106

    Figure Lengend Snippet: Architecture of the DNA–primase complex. ( A ) Crystal structure of the catalytic domain of RepB′ in complex with ssiA (3′Δ13). Catalytic domain in ribbon representation is green, and ssiA (3′Δ13) is shown as

    Article Snippet: The primase assay was performed in NEB buffer 2 [50 mM NaCl, 10 mM Tris·HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT] and incubated at 37 °C and 72 °C for 10 min.

    Techniques:

    Comparison of catalytic centers of Pfu primase ( A ) and RepB′ ( B ). Superposition of RepB′ on Pfu (Protein Data Bank ID code 1G71 ) yields a Z-score of 9.9 and an rmsd of 2.8 Å for 148 C α positions. The catalytic cores of

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

    Article Title: Structure and function of primase RepB? encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

    doi: 10.1073/pnas.0902910106

    Figure Lengend Snippet: Comparison of catalytic centers of Pfu primase ( A ) and RepB′ ( B ). Superposition of RepB′ on Pfu (Protein Data Bank ID code 1G71 ) yields a Z-score of 9.9 and an rmsd of 2.8 Å for 148 C α positions. The catalytic cores of

    Article Snippet: The primase assay was performed in NEB buffer 2 [50 mM NaCl, 10 mM Tris·HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT] and incubated at 37 °C and 72 °C for 10 min.

    Techniques:

    Complementary strand synthesis initiated by RepB′. ( A ) Principle of primase assay. A complete reaction mixture contains circular ssM13 DNA carrying ssiA , primase RepB′, Vent-DNA polymerase, and dNTPs. RepB′ synthesizes a primer

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

    Article Title: Structure and function of primase RepB? encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

    doi: 10.1073/pnas.0902910106

    Figure Lengend Snippet: Complementary strand synthesis initiated by RepB′. ( A ) Principle of primase assay. A complete reaction mixture contains circular ssM13 DNA carrying ssiA , primase RepB′, Vent-DNA polymerase, and dNTPs. RepB′ synthesizes a primer

    Article Snippet: The primase assay was performed in NEB buffer 2 [50 mM NaCl, 10 mM Tris·HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT] and incubated at 37 °C and 72 °C for 10 min.

    Techniques:

    Complementation assay is shown for the catalytic and helix-bundle domain (see the legend for B ). Cat + helix-b. domain, isolated catalytic and helix bundle domains in molar ratio of 1:1. Catalytic domain, primase assay in the presence of the catalytic

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

    Article Title: Structure and function of primase RepB? encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

    doi: 10.1073/pnas.0902910106

    Figure Lengend Snippet: Complementation assay is shown for the catalytic and helix-bundle domain (see the legend for B ). Cat + helix-b. domain, isolated catalytic and helix bundle domains in molar ratio of 1:1. Catalytic domain, primase assay in the presence of the catalytic

    Article Snippet: The primase assay was performed in NEB buffer 2 [50 mM NaCl, 10 mM Tris·HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT] and incubated at 37 °C and 72 °C for 10 min.

    Techniques: Isolation

    Initiation of SV40 DNA replication in vitro by DNA polymerase α-primase (pol-prim) prephosphorylated by cyclin A/cdk2 or cyclin E/cdk2. (A) Increasing amounts of the indicated preparations of DNA polymerase α-primase were tested for initiation of SV40 DNA replication. The initiation products of each reaction were separated by denaturing PAGE and quantitated by PhosphorImager analysis, and the background (data not shown) was subtracted to give the number of SV40 initiation units per microliter of DNA polymerase α-primase. The bar to the right indicates primers of 8 to 10 nucleotides. (B) The SV40 initiation activity in each reaction in A was divided by the number of primase units per microliter (data not shown) determined for the corresponding preparation of phosphorylated DNA polymerase α-primase. The bar graph shows the average relative initiation activity of each preparation; that of the mock-phosphorylated preparation was set to 100%. The error bars represent the variation obtained from three different reactions with each preparation of DNA polymerase α-primase.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Dependent Regulation of Human DNA Polymerase ?-Primase Activity by Phosphorylation

    doi:

    Figure Lengend Snippet: Initiation of SV40 DNA replication in vitro by DNA polymerase α-primase (pol-prim) prephosphorylated by cyclin A/cdk2 or cyclin E/cdk2. (A) Increasing amounts of the indicated preparations of DNA polymerase α-primase were tested for initiation of SV40 DNA replication. The initiation products of each reaction were separated by denaturing PAGE and quantitated by PhosphorImager analysis, and the background (data not shown) was subtracted to give the number of SV40 initiation units per microliter of DNA polymerase α-primase. The bar to the right indicates primers of 8 to 10 nucleotides. (B) The SV40 initiation activity in each reaction in A was divided by the number of primase units per microliter (data not shown) determined for the corresponding preparation of phosphorylated DNA polymerase α-primase. The bar graph shows the average relative initiation activity of each preparation; that of the mock-phosphorylated preparation was set to 100%. The error bars represent the variation obtained from three different reactions with each preparation of DNA polymerase α-primase.

    Article Snippet: Phosphatase reactions were carried out by incubating DNA polymerase α-primase that was either in solution or immobilized on SJK 132-20–Sepharose beads with 500 U of λ-phosphatase (New England Biolabs, Beverly, Mass.) in phosphatase buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.01% Nonidet P-40) for 1 h at 30°C.

    Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Activity Assay

    Cyclin A-dependent kinases induce a p68 mobility shift which is reversible with λ-phosphatase. (A) Purified recombinant DNA polymerase α-primase (2 μg) was incubated without (−) or with increasing amounts (200, 400, and 600 pmol/h) of cyclin/cdk kinases for 20 min. The subunits of DNA polymerase α-primase were separated by SDS–7.5% PAGE, and the p68 subunit was detected by Western blotting using monoclonal antibody 9D5. (B) Two micrograms of DNA polymerase α-primase, immobilized on SJK 132-20–Sepharose, was prephosphorylated with the indicated cyclin/cdk complexes or mock phosphorylated (mock), washed with phosphate-buffered saline, and incubated with (+) λ-phosphatase (λ-PPase) in the presence (+) or absence (−) of phosphatase inhibitors, as indicated, or without phosphatase (−). The p68 bands were separated by SDS-PAGE and detected by immunoblotting with monoclonal antibody 9D5. The values on the right are molecular sizes in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Dependent Regulation of Human DNA Polymerase ?-Primase Activity by Phosphorylation

    doi:

    Figure Lengend Snippet: Cyclin A-dependent kinases induce a p68 mobility shift which is reversible with λ-phosphatase. (A) Purified recombinant DNA polymerase α-primase (2 μg) was incubated without (−) or with increasing amounts (200, 400, and 600 pmol/h) of cyclin/cdk kinases for 20 min. The subunits of DNA polymerase α-primase were separated by SDS–7.5% PAGE, and the p68 subunit was detected by Western blotting using monoclonal antibody 9D5. (B) Two micrograms of DNA polymerase α-primase, immobilized on SJK 132-20–Sepharose, was prephosphorylated with the indicated cyclin/cdk complexes or mock phosphorylated (mock), washed with phosphate-buffered saline, and incubated with (+) λ-phosphatase (λ-PPase) in the presence (+) or absence (−) of phosphatase inhibitors, as indicated, or without phosphatase (−). The p68 bands were separated by SDS-PAGE and detected by immunoblotting with monoclonal antibody 9D5. The values on the right are molecular sizes in kilodaltons.

    Article Snippet: Phosphatase reactions were carried out by incubating DNA polymerase α-primase that was either in solution or immobilized on SJK 132-20–Sepharose beads with 500 U of λ-phosphatase (New England Biolabs, Beverly, Mass.) in phosphatase buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.01% Nonidet P-40) for 1 h at 30°C.

    Techniques: Mobility Shift, Purification, Recombinant, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page

    The SV40 initiation activity of DNA polymerase α-primase (pol-prim) containing the 4× alanine mutant form of p68 is resistant to inhibition by cyclin A/cdk2. Prephosphorylated and mock-phosphorylated wt (A) and mutant (C) forms of DNA polymerase α-primase were assayed for the ability to initiate SV40 DNA replication. The reaction products were separated on 20% urea gels as described in Materials and Methods and analyzed by PhosphorImager. A control reaction was carried out in the absence of SV40 T antigen (−T). The SV40 initiation activities of the mock- and cyclin A/cdk2-phosphorylated forms of DNA polymerase α-primase (units per microliter) were divided by the number of primase units per microliter of the corresponding preparation. The bar graphs show the SV40 initiation activities of the cyclin A/cdk2-phosphorylated wt (B) and mutant (D) forms of DNA polymerase α-primase relative to the activity of the corresponding mock-phosphorylated preparation, which was set to 100%. Error bars indicate the variation observed in three different reactions with each preparation of DNA polymerase α-primase.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Dependent Regulation of Human DNA Polymerase ?-Primase Activity by Phosphorylation

    doi:

    Figure Lengend Snippet: The SV40 initiation activity of DNA polymerase α-primase (pol-prim) containing the 4× alanine mutant form of p68 is resistant to inhibition by cyclin A/cdk2. Prephosphorylated and mock-phosphorylated wt (A) and mutant (C) forms of DNA polymerase α-primase were assayed for the ability to initiate SV40 DNA replication. The reaction products were separated on 20% urea gels as described in Materials and Methods and analyzed by PhosphorImager. A control reaction was carried out in the absence of SV40 T antigen (−T). The SV40 initiation activities of the mock- and cyclin A/cdk2-phosphorylated forms of DNA polymerase α-primase (units per microliter) were divided by the number of primase units per microliter of the corresponding preparation. The bar graphs show the SV40 initiation activities of the cyclin A/cdk2-phosphorylated wt (B) and mutant (D) forms of DNA polymerase α-primase relative to the activity of the corresponding mock-phosphorylated preparation, which was set to 100%. Error bars indicate the variation observed in three different reactions with each preparation of DNA polymerase α-primase.

    Article Snippet: Phosphatase reactions were carried out by incubating DNA polymerase α-primase that was either in solution or immobilized on SJK 132-20–Sepharose beads with 500 U of λ-phosphatase (New England Biolabs, Beverly, Mass.) in phosphatase buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.01% Nonidet P-40) for 1 h at 30°C.

    Techniques: Activity Assay, Mutagenesis, Inhibition