∘ user enzyme  (New England Biolabs)


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    Name:
    USER Enzyme
    Description:
    USER Enzyme 250 units
    Catalog Number:
    m5505l
    Price:
    297
    Size:
    250 units
    Category:
    Other Enzymes
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    Structured Review

    New England Biolabs ∘ user enzyme
    USER Enzyme
    USER Enzyme 250 units
    https://www.bioz.com/result/∘ user enzyme/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    ∘ user enzyme - by Bioz Stars, 2020-07
    99/100 stars

    Related Products / Commonly Used Together

    ∘ userbstbi-compatible digested pms26
    general materials

    Images

    1) Product Images from "A versatile element for gene addition in bacterial chromosomes"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1085

    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 ).
    Figure Legend 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 ).

    Techniques Used: Clone Assay, Sequencing, Construct

    Related Articles

    Clone Assay:

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: .. Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously . ..

    Amplification:

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach
    Article Snippet: .. Step 7 To generate cohesive ends, the amplified PCR product was treated with the USER enzyme (New England Biolabs) according to the manufacturer's instructions, which specifically removes deoxyuridine in the DNA. .. The final product contains unique 7 bp and 4 bp overhangs at each end, which were used to facilitate cloning into the lentiviral vector pLL3.7 (kindly provided by Dr. Luk Van Parijs).

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    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. .. For duplex sequencing, 10 µl purified, adapter ligated library of insert 3 were incubated with 1 U USER enzyme in 1× NEB CS Buffer in a reaction volume of 20 µl at 37 °C for 1h.

    Magnetic Beads:

    Article Title: AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq
    Article Snippet: .. The following reagents are required for this section of program, as specified in the instrument setup ( ): 100% isopropanol, EB (10 mM Tris-HCl pH 8), freshly prepared 85% ethanol (all on the deck at room temperature), proteinase K 20 mg/ml (Thermo Fisher, EO0491), glycogen 20 mg/mg (Thermo Fisher, R0561), carboxylated magnetic beads (Invitrogen, 65011), PCR mix (NEBNext Ultra II Q5 Master mix, NEB M0544), USER enzyme (NEB M5505), all placed in 1.5 ml conical tubes in a cold Peltier block. .. Ampure XP (Beckman Coulter, A63881) are thoroughly mixed and aliquoted column-wise according to the pattern of “Sample Plate 2” in a 96-well storage plate (AB0765, Thermo Fisher), using 100 µl of beads per well.

    Construct:

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    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. .. For duplex sequencing, 10 µl purified, adapter ligated library of insert 3 were incubated with 1 U USER enzyme in 1× NEB CS Buffer in a reaction volume of 20 µl at 37 °C for 1h.

    Purification:

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

    Incubation:

    Article Title: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
    Article Snippet: .. Following protein expression, 5 μL of lysate was combined with 35 μL of ssDNA (1.8 μM) and USER enzyme (1 unit) in CutSmart buffer (New England Biolabs) (50 mM potassium acetate, 29 mM Tris-acetate, 10 mM magnesium acetate, 100 ug/mL BSA, pH 7.9) and incubated at 37 °C for 2 h. Cleaved U-containing substrates were resolved from full-length unmodified substrates on a 10% TBE-urea gel (Bio-Rad). .. Expression and purification of His6 -rAPOBEC1-linker-dCas9 fusions E. coli BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding pET28b-His6 -rAPOBEC-linker-dCas9 with GGS, (GGS)3 , XTEN, or (GGS)7 linkers.

    Blocking Assay:

    Article Title: AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq
    Article Snippet: .. The following reagents are required for this section of program, as specified in the instrument setup ( ): 100% isopropanol, EB (10 mM Tris-HCl pH 8), freshly prepared 85% ethanol (all on the deck at room temperature), proteinase K 20 mg/ml (Thermo Fisher, EO0491), glycogen 20 mg/mg (Thermo Fisher, R0561), carboxylated magnetic beads (Invitrogen, 65011), PCR mix (NEBNext Ultra II Q5 Master mix, NEB M0544), USER enzyme (NEB M5505), all placed in 1.5 ml conical tubes in a cold Peltier block. .. Ampure XP (Beckman Coulter, A63881) are thoroughly mixed and aliquoted column-wise according to the pattern of “Sample Plate 2” in a 96-well storage plate (AB0765, Thermo Fisher), using 100 µl of beads per well.

    Expressing:

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: .. Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously . ..

    Article Title: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
    Article Snippet: .. Following protein expression, 5 μL of lysate was combined with 35 μL of ssDNA (1.8 μM) and USER enzyme (1 unit) in CutSmart buffer (New England Biolabs) (50 mM potassium acetate, 29 mM Tris-acetate, 10 mM magnesium acetate, 100 ug/mL BSA, pH 7.9) and incubated at 37 °C for 2 h. Cleaved U-containing substrates were resolved from full-length unmodified substrates on a 10% TBE-urea gel (Bio-Rad). .. Expression and purification of His6 -rAPOBEC1-linker-dCas9 fusions E. coli BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding pET28b-His6 -rAPOBEC-linker-dCas9 with GGS, (GGS)3 , XTEN, or (GGS)7 linkers.

    Polymerase Chain Reaction:

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach
    Article Snippet: .. Step 7 To generate cohesive ends, the amplified PCR product was treated with the USER enzyme (New England Biolabs) according to the manufacturer's instructions, which specifically removes deoxyuridine in the DNA. .. The final product contains unique 7 bp and 4 bp overhangs at each end, which were used to facilitate cloning into the lentiviral vector pLL3.7 (kindly provided by Dr. Luk Van Parijs).

    Article Title: AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq
    Article Snippet: .. The following reagents are required for this section of program, as specified in the instrument setup ( ): 100% isopropanol, EB (10 mM Tris-HCl pH 8), freshly prepared 85% ethanol (all on the deck at room temperature), proteinase K 20 mg/ml (Thermo Fisher, EO0491), glycogen 20 mg/mg (Thermo Fisher, R0561), carboxylated magnetic beads (Invitrogen, 65011), PCR mix (NEBNext Ultra II Q5 Master mix, NEB M0544), USER enzyme (NEB M5505), all placed in 1.5 ml conical tubes in a cold Peltier block. .. Ampure XP (Beckman Coulter, A63881) are thoroughly mixed and aliquoted column-wise according to the pattern of “Sample Plate 2” in a 96-well storage plate (AB0765, Thermo Fisher), using 100 µl of beads per well.

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

    Plasmid Preparation:

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: .. Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously . ..

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  • 99
    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 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/user enzyme/product/New England Biolabs
    Average 99 stars, based on 965 article reviews
    Price from $9.99 to $1999.99
    user enzyme - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

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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

    Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Journal: Nature Communications

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    doi: 10.1038/s41467-018-04870-w

    Figure Lengend Snippet: Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Article Snippet: Subsequent solid-phase separation using streptavidin-coated magnetic beads removes unbound precursors, and applying USER enzyme mix (New England Biolabs) cleaves the oligo products from the tags at the dU site.

    Techniques: Purification, Synthesized, Sequencing, Concentration Assay, Oligo Synthesis, Magnetic Beads, Next-Generation Sequencing, Standard Deviation, Binding Assay

    Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Modification, High Throughput Screening Assay, DNA Sequencing, Amplification, Transfection, Expressing, Plasmid Preparation

    Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Binding Assay, Activity Assay, Standard Deviation

    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