t5 exonuclease  (New England Biolabs)


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  • 99
    Name:
    T5 Exonuclease
    Description:

    Catalog Number:
    M0363L
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs t5 exonuclease

    https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease - by Bioz Stars, 2019-10
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: One milliliter of 5X TEDA solution contained 0.5 M Tris–HCl pH 7.5, 50 mM MgCl2 , 50 mM dithiothreitol, 0.25 g of PEG 8000, and 1 μl of 10 U/μl T5 exonuclease (New England Biolab, Beijing). .. One milliliter of 5X TEDA solution contained 0.5 M Tris–HCl pH 7.5, 50 mM MgCl2 , 50 mM dithiothreitol, 0.25 g of PEG 8000, and 1 μl of 10 U/μl T5 exonuclease (New England Biolab, Beijing).

    Transfection:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Higher yields can be obtained if the T5 exonuclease treatment is performed directly in the second strand synthesis reaction ( ). .. We find that treatment with T5 exonuclease does not improve transfection efficiencies, and EGFP constructs treated or not treated with the enzyme result in similar efficiencies ( ). .. We also found that bacterial EGFP plasmid purified using the same method and of identical purity results in higher efficiencies than both types of constructs, likely due to differences in the plasmid coiling.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Incubation:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Polymerization reactions starting with 80 μg of ssDNA annealed with ODN were carried out in 1X NEBuffer 2 (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, pH 7.9), containing 7.5% PEG-8000, 50 μg/mL BSA, 600 μM each dNTP, 1 mM ATP, 80 U T4 DNA ligase, and 40 U T4 DNA polymerase, in a total volume of 600 μL. .. In order to enzymatically digest nicked, linear and single-stranded DNA, T5 exonuclease (New England Biolabs, Cat. #M0363) was added for the samples indicated in the text, directly to the second strand synthesis reaction or to purified dsDNA product in buffer NEBuffer 2 at 5 units per μg of starting ssDNA or dsDNA, respectively, and incubated for an hour at 37°C. .. Reactions were stopped by the addition of EDTA to a final concentration of 11 μM and DNA purified.

    Construct:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The faint upper band in the ssDNA preparation has the same migration pattern as M13KO7 ssDNA. .. While we do not observe significant M13KO7 ssDNA contamination in purified constructs not treated with T5 exonuclease , treatment with T5 exonuclease can be employed if minimizing ssDNA contamination is preferred. (TIF) Click here for additional data file. .. S1 Table Sequences of oligodeoxynucleotides containing 5’ phosphorylation (P), 8-oxoguanine (8-oxoG), 5-hydroxyuracil (5-OHU), or dihydrouracil (DHU), used for second strand synthesis. (TIF) Click here for additional data file.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Higher yields can be obtained if the T5 exonuclease treatment is performed directly in the second strand synthesis reaction ( ). .. We find that treatment with T5 exonuclease does not improve transfection efficiencies, and EGFP constructs treated or not treated with the enzyme result in similar efficiencies ( ). .. We also found that bacterial EGFP plasmid purified using the same method and of identical purity results in higher efficiencies than both types of constructs, likely due to differences in the plasmid coiling.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Purification:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The faint upper band in the ssDNA preparation has the same migration pattern as M13KO7 ssDNA. .. While we do not observe significant M13KO7 ssDNA contamination in purified constructs not treated with T5 exonuclease , treatment with T5 exonuclease can be employed if minimizing ssDNA contamination is preferred. (TIF) Click here for additional data file. .. S1 Table Sequences of oligodeoxynucleotides containing 5’ phosphorylation (P), 8-oxoguanine (8-oxoG), 5-hydroxyuracil (5-OHU), or dihydrouracil (DHU), used for second strand synthesis. (TIF) Click here for additional data file.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: Polymerization reactions starting with 80 μg of ssDNA annealed with ODN were carried out in 1X NEBuffer 2 (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, pH 7.9), containing 7.5% PEG-8000, 50 μg/mL BSA, 600 μM each dNTP, 1 mM ATP, 80 U T4 DNA ligase, and 40 U T4 DNA polymerase, in a total volume of 600 μL. .. In order to enzymatically digest nicked, linear and single-stranded DNA, T5 exonuclease (New England Biolabs, Cat. #M0363) was added for the samples indicated in the text, directly to the second strand synthesis reaction or to purified dsDNA product in buffer NEBuffer 2 at 5 units per μg of starting ssDNA or dsDNA, respectively, and incubated for an hour at 37°C. .. Reactions were stopped by the addition of EDTA to a final concentration of 11 μM and DNA purified.

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

    Concentration Assay:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: However, the supplier claims that T5 exonuclease is not inactivated by heating ( https://www.neb.com ). .. Our results showed that inactivation of T5 exonuclease is not required as long as the concentration of the enzyme is low, consistent with the heat-resistant property of the enzyme. .. The low temperature may also facilitate annealing of short overhangs.

    Generated:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: Further, without a thermophilic DNA polymerase that requires high temperature for activity, TEDA allows the reaction at 30°C rather than 50°C as in the Gibson and Hot Fusion methods ( ). .. As reported, 50°C keeps Phusion DNA polymerase and Taq DNA ligase active as well as inactivating T5 exonuclease after its has generated the 5′-single strand ends ( ). .. However, the supplier claims that T5 exonuclease is not inactivated by heating ( https://www.neb.com ).

    other:

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site
    Article Snippet: Nt.Bbv CI nicking endonuclease, Eco NI, Fpg, hOGG1, UDG, APE1, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA, USA).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: For optimization, PEG 8000 and the proper dilution of T5 exonuclease were two key factors for TEDA (Figure ).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: However, both T5 exonuclease and T4 DNA polymerase are semi-processive exonucleases ( , ).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: TEDA uses only T5 exonuclease to create 3′-overhangs of linear DNA fragments, which anneal to form circle DNA with gaps.

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: TEDA requires 0.04–0.08 U T5 exonuclease per reaction ( ).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: However, the supplier claims that T5 exonuclease is not inactivated by heating ( https://www.neb.com ).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: The simplified method with only T5 exonuclease worked well at a similar efficiency to the Gibson method and the Hot Fusion method (Figure and ).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: The optimal reaction was at 30°C for 40 min with 0.04– 0.08 U T5 exonuclease per reaction ( –C).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: TEDA uses T5 exonuclease that generates 3′-overhangs.

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: T5 exonuclease is not only a very efficient exonuclease , but also a flap endonuclease , capable of threading 5′-overhangs and cleaving them ( ).

    Polymerase Chain Reaction:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: T5 exonuclease (New England Biolab, Beijing) was used in Gibson, Hot Fusion, and TEDA. .. T5 exonuclease (New England Biolab, Beijing) was used in Gibson, Hot Fusion, and TEDA.

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: One milliliter of 5X TEDA solution contained 0.5 M Tris–HCl pH 7.5, 50 mM MgCl2 , 50 mM dithiothreitol, 0.25 g of PEG 8000, and 1 μl of 10 U/μl T5 exonuclease (New England Biolab, Beijing). .. One milliliter of 5X TEDA solution contained 0.5 M Tris–HCl pH 7.5, 50 mM MgCl2 , 50 mM dithiothreitol, 0.25 g of PEG 8000, and 1 μl of 10 U/μl T5 exonuclease (New England Biolab, Beijing).

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: Most recently, Huang and colleagues further optimized this method and offered a low-cost solution for DNA assembly ( ). .. When the QuikChangeTM PCR product was treated with T5 exonuclease according to TEDA, the efficiency of forming positive colonies was 10-fold higher than without T5 exonuclease treatment (Figure ). .. Further, the number of colonies formed by using TEDA for two-fragment assembly were 50 to 500 fold higher than co-transformation of the same amount of linearized vector and insert directly into competent cells, which were used as negative control as reported in several figures (Figure , ).

    Plasmid Preparation:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: The presence of nicked and linear product could affect transfection efficiencies. .. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ , ], and EGFP bacterial maxiprep. .. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product , albeit at the cost of reduction in yield.

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  • 99
    New England Biolabs t5 exonuclease
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

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