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standard restriction cloning with ndei  (New England Biolabs)


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    New England Biolabs standard restriction cloning with ndei
    Standard Restriction Cloning With Ndei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4822 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/standard restriction cloning with ndei/product/New England Biolabs
    Average 99 stars, based on 4822 article reviews
    standard restriction cloning with ndei - by Bioz Stars, 2026-06
    99/100 stars

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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