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restriction endonuclease cloning workflows  (New England Biolabs)


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    New England Biolabs restriction endonuclease cloning workflows
    Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.
    Restriction Endonuclease Cloning Workflows, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 33839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/restriction+endonuclease+cloning+workflows/T4+DNA+Ligase/pmc12958811-141-8-38
    Average 99 stars, based on 33839 article reviews
    restriction endonuclease cloning workflows - by Bioz Stars, 2026-11
    99/100 stars

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    1) Product Images from "Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis"

    Article Title: Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis

    Journal: G3: Genes | Genomes | Genetics

    doi: 10.1093/g3journal/jkag006

    Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.
    Figure Legend Snippet: Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.

    Techniques Used: Plasmid Preparation, Diagnostic Assay, Agarose Gel Electrophoresis, Biomarker Discovery, Construct

    Related Articles

    Cloning:

    Article Title: Deep-mutational scanning libraries using tiled-region exchange mutagenesis
    Article Snippet: .. While this is a common occurrence in some restriction-endonuclease cloning workflows, it was not expected to occur based on the measured fidelity of T4 DNA ligase (5-GGAA ligated to 3-CCCT was seen approximately 0.1% of the time in NEB’s screen( )). ..

    Article Title: Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis
    Article Snippet: .. While this is a common occurrence in some restriction-endonuclease cloning workflows, it was not expected to occur based on the measured fidelity of T4 DNA ligase (5-GGAA ligated to 3-CCCT was seen approximately 0.1% of the time in NEB's screen [ ]). ..



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    restriction endonuclease cloning workflows  (New England Biolabs)
    99
    New England Biolabs restriction endonuclease cloning workflows
    Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.
    Restriction Endonuclease Cloning Workflows, 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
    https://www.bioz.com/product/restriction+endonuclease+cloning+workflows/T4+DNA+Ligase/pmc12958811-141-8-38
    Average 99 stars, based on 1 article reviews
    restriction endonuclease cloning workflows - by Bioz Stars, 2026-11
    99/100 stars
    		  Buy from Supplier

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    Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.

    Journal: G3: Genes | Genomes | Genetics

    Article Title: Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis

    doi: 10.1093/g3journal/jkag006

    Figure Lengend Snippet: Optimization of Bxb1 integrase-mediated fusion between a linear R6K (oriγ) recipient vector and a donor fragment. A complete fusion reaction yields 4 diagnostic bands corresponding to the linear R6K backbone, the donor fragment, and the 2 recombination products, and is consistent across each reaction. a) Agarose gel of temperature series. Lane 1: DNA size ladder. Lanes 2 to 5: fusion reactions performed at 20 °C, 25 °C, 30 °C, and 37 °C, respectively, for 1 h. b) Additive series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 2 mM spermidine, 200 µg/ml BSA, 5% PEG, and 10% propylene glycol, respectively. Reaction at 37 °C for 1 h. c) Salt tolerance series. Lane 1: DNA ladder. Lanes 2 to 5: reactions supplemented with 25 mM, 50, 75, and 100 mM NaCl, respectively. Reaction at 37 °C for 1 h. d) Validation of plasmid fusions using PmeI restriction endonuclease, which confirms correct linearization of fragments. Lane 1: 10 kb DNA ladder; Lane 2: PmeI digested barcode plasmid before Bxb1 fusion reaction; Lane 3: PmeI digested mutagenized plasmid before Bxb1 fusion reaction; Lane 4: PmeI digested fusion construct of barcode plasmid and mutagenized plasmid after Bxb1 fusion reaction. Band sizes are consistent with PmeI restriction site locations.

    Article Snippet: While this is a common occurrence in some restriction-endonuclease cloning workflows, it was not expected to occur based on the measured fidelity of T4 DNA ligase (5-GGAA ligated to 3-CCCT was seen approximately 0.1% of the time in NEB's screen [ ]).

    Techniques: Plasmid Preparation, Diagnostic Assay, Agarose Gel Electrophoresis, Biomarker Discovery, Construct