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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    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 969 article reviews
    Price from $9.99 to $1999.99
    user enzyme - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications"

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

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

    doi: 10.1093/dnares/dsw038

    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.
    Figure Legend 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.

    Techniques Used: Amplification, Construct

    2) Product Images from "Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity"

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

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1793

    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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Activity Assay, Standard Deviation

    3) 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

    4) Product Images from "Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach"

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007029

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.
    Figure Legend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Techniques Used: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing

    5) Product Images from "Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications"

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

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

    doi: 10.1093/dnares/dsw038

    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.
    Figure Legend 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.

    Techniques Used: Amplification, Construct

    6) Product Images from "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage"

    Article Title: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

    Journal: Nature

    doi: 10.1038/nature17946

    Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Activity Assay, Derivative Assay, In Vitro, Incubation, Positive Control, Sequencing, Staining, Polyacrylamide Gel Electrophoresis, Purification, Isolation

    7) Product Images from "AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq"

    Article Title: AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

    Journal: bioRxiv

    doi: 10.1101/2020.03.30.016287

    AutoRELACS workflow ensures comprehensive integration of RELACS barcodes a) Overview of AutoRELACS protocol. 1-M) Nuclei of formaldehyde-fixed cells are extracted manually using adjusted ultrasound ( 14 ). The nuclear envelope is permeabilized, and the chromatin digested in situ using a 4-cutter restriction enzyme (RE). 2-A) Digested chromatin from each sample is automatically barcoded. Upon completion, the liquid handler pools all barcoded samples into a unique tube (Biomek i7 program: “RELACS_Barcoding”). 3-M) Pooled samples are collected by the user and nuclei are lysed using focused sonication. 4-A) The barcoded chromatin is aliquoted according to the number of required immunoprecipitation (IP) reactions into corresponding ChIP reaction mixes. The ChIP reactions are carried out overnight in parallel at room temperature on the Biomek i7 workstation. Upon completion, the ChIP-ped chromatin is sequestrated using beads and automatically washed 4 times at increasing stringency conditions and finally eluted in the elution buffer (Biomek program: “RELACS_ChIP_Elution”). 5-A) Subsequently, the eluted chromatin is decrosslinked and the DNA is purified. DNA is amplified via PCR using primers carrying Illumina dual indexes. Optionally, the liquid handler performs multiple rounds of purification and size selection using Ampure XP beads (Biomek program: “RELACS_Decrosslink_FinalLibraries”). A: Automated; M: Manual. 6-A) Libraries are sequenced on Illumina’s sequencing devices. Upon completion of the sequencing run, bcl2 files are automatically converted to fastq format and input into the fully automated ChIP-seq workflow available as part of the snakePipes suite ( 13 ). SnakePipes’ ChIP-seq workflow performs demultiplexing of reads on RELACS custom barcodes, quality controls, mapping and filtering of duplicate reads using unique molecular identifiers (UMI), and further downstream analysis like generation of input-normalized coverage tracks and peak calling. b) Distribution of RELACS barcodes in two independent input chromatin pools. 60 barcodes are integrated into the digested chromatin of two independent batches of S2 cells. Sequencing of the input chromatin pool for replicate 1 (upper panel) and replicate 2 (lower panel), reveals the percentage of input reads for each barcode used (y-axis). The ideal uniform distribution (100/60) is represented as a dotted line. The shaded gray area shows one standard deviation from the mean of the observed distribution.
    Figure Legend Snippet: AutoRELACS workflow ensures comprehensive integration of RELACS barcodes a) Overview of AutoRELACS protocol. 1-M) Nuclei of formaldehyde-fixed cells are extracted manually using adjusted ultrasound ( 14 ). The nuclear envelope is permeabilized, and the chromatin digested in situ using a 4-cutter restriction enzyme (RE). 2-A) Digested chromatin from each sample is automatically barcoded. Upon completion, the liquid handler pools all barcoded samples into a unique tube (Biomek i7 program: “RELACS_Barcoding”). 3-M) Pooled samples are collected by the user and nuclei are lysed using focused sonication. 4-A) The barcoded chromatin is aliquoted according to the number of required immunoprecipitation (IP) reactions into corresponding ChIP reaction mixes. The ChIP reactions are carried out overnight in parallel at room temperature on the Biomek i7 workstation. Upon completion, the ChIP-ped chromatin is sequestrated using beads and automatically washed 4 times at increasing stringency conditions and finally eluted in the elution buffer (Biomek program: “RELACS_ChIP_Elution”). 5-A) Subsequently, the eluted chromatin is decrosslinked and the DNA is purified. DNA is amplified via PCR using primers carrying Illumina dual indexes. Optionally, the liquid handler performs multiple rounds of purification and size selection using Ampure XP beads (Biomek program: “RELACS_Decrosslink_FinalLibraries”). A: Automated; M: Manual. 6-A) Libraries are sequenced on Illumina’s sequencing devices. Upon completion of the sequencing run, bcl2 files are automatically converted to fastq format and input into the fully automated ChIP-seq workflow available as part of the snakePipes suite ( 13 ). SnakePipes’ ChIP-seq workflow performs demultiplexing of reads on RELACS custom barcodes, quality controls, mapping and filtering of duplicate reads using unique molecular identifiers (UMI), and further downstream analysis like generation of input-normalized coverage tracks and peak calling. b) Distribution of RELACS barcodes in two independent input chromatin pools. 60 barcodes are integrated into the digested chromatin of two independent batches of S2 cells. Sequencing of the input chromatin pool for replicate 1 (upper panel) and replicate 2 (lower panel), reveals the percentage of input reads for each barcode used (y-axis). The ideal uniform distribution (100/60) is represented as a dotted line. The shaded gray area shows one standard deviation from the mean of the observed distribution.

    Techniques Used: In Situ, Sonication, Immunoprecipitation, Chromatin Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Selection, Sequencing, Standard Deviation

    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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    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 952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/user enzyme/product/New England Biolabs
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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

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Journal: PLoS ONE

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    doi: 10.1371/journal.pone.0007029

    Figure Lengend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    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.

    Techniques: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing