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    New England Biolabs user enzyme
    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; <t>HSI_insert_1</t> construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without <t>USER</t> treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.
    User Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    doi: 10.1093/dnares/dsw038

    Figure Lengend Snippet: Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Article Snippet: Treatments with the USER enzyme were performed on HSI_insert_1 by incubating 2 × 107 copies HSI_insert construct with 1 U USER enzyme (NEB) in 1× Phusion HF Buffer in a reaction volume of 20 µl at 37 °C for 30 min prior to amplification.

    Techniques: Amplification, Construct

    Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Journal: Nucleic Acids Research

    Article Title: A versatile element for gene addition in bacterial chromosomes

    doi: 10.1093/nar/gkr1085

    Figure Lengend Snippet: Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Article Snippet: General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions.

    Techniques: Clone Assay, Sequencing, Construct