user  (New England Biolabs)


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  • 97
    Name:
    USER Enzyme
    Description:

    Catalog Number:
    M5505S
    Price:
    None
    Score:
    85
    Buy from Supplier
    Name:
    USER Enzyme
    Description:
    USER Enzyme 250 units
    Catalog Number:
    M5505L
    Price:
    292
    Size:
    250 units
    Category:
    Other Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    New England Biolabs user
    USER Enzyme
    USER Enzyme 250 units
    https://www.bioz.com/result/user/product/New England Biolabs
    Average 97 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    user - by Bioz Stars, 2019-12
    97/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: Paragraph title: General USER cloning ... For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: A plasmid encoding the human codon-optimized Streptococcus pyogenes Cas9 nuclease with an NLS and 3×FLAG tag (Addgene plasmid 43861) was used as the wild-type Cas9 expression plasmid. .. 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 . .. Plasmid constructs generated in this work will be deposited with Addgene.

    Amplification:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Next, the backbone of the bacterial base editor plasmid template , was amplified with primers NMG-799 and NMG-824 ( ) and Phusion U Green Multiplex PCR Master Mix (100 μL per well in a 98-well PCR plate, 5–6 plates total, Tm 66 °C, 4.5-min extension) following the manufacturer’s protocol. .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Second-strand cDNA was then bulk amplified with LongAmp HotStart Taq using Pac_for_dU and Pac_rev_dU primers, which contain an internal 5′-dUTP. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The amplified amplicons were purified by ethanol precipitation and re-purified with Qiaquick PCR purification columns (Qiagen). .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Article Title: Genome-wide profiling reveals cancer-related genes with switched alternative polyadenylation sites in colorectal cancer
    Article Snippet: Next, Illumina p5/p7 adaptors were ligated to the released cDNA. .. Before PCR amplification, the second strand of cDNA which had dUTP was digested by USER (NEB) to achieve chain specificity sequencing. .. Finally, a series of 3T-seq libraries were sequenced by Illumina Hiseq X Ten.

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: First, 15–20 µg of the purified amplicon were mixed with 50 units of lambda exonuclease (New England Biolabs) in a 150 µl reaction containing 1 × lambda exonuclease buffer and incubated for 1 h at 37 °C, to remove the bottom strand. .. Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C.

    Article Title: Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes
    Article Snippet: The cDNA templates were purified using a Qiagen kit (Venlo, Netherlands) followed by end repair, poly A tailing, and adaptor connection. .. The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ]. .. Finally, the library was sequenced using an IlluminaHiSeq 2000.

    Article Title: Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX
    Article Snippet: In the first experimental procedure, aDNA extracts were treated with USER (New England Biolabs), a commercial DNA repair mix containing Uracil DNA Glycosylase (UNG) and Endonuclease VIII (EndoVIII) (method 1-USER, orange figure header backgrounds). .. In the first experimental procedure, aDNA extracts were treated with USER (New England Biolabs), a commercial DNA repair mix containing Uracil DNA Glycosylase (UNG) and Endonuclease VIII (EndoVIII) (method 1-USER, orange figure header backgrounds).

    Synthesized:

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: Libraries of oligonucleotides (~150 nt) were synthesized by ink-jet printing on programmable microarrays (Agilent Technologies) and released to form a combined library of 330,000 oligonucleotides. .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Briefly, after RNA fragmentation, double-stranded cDNA was synthesized by replacing dTTPs (deoxythymidine triphosphate) with dUTPs (deoxyuridine triphosphate) in reaction buffer used for second strand cDNA synthesis. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Genome-wide profiling reveals cancer-related genes with switched alternative polyadenylation sites in colorectal cancer
    Article Snippet: When the second strand of cDNA chain was synthesized, dNTP mixture contained dUTP instead of dTTP. .. Before PCR amplification, the second strand of cDNA which had dUTP was digested by USER (NEB) to achieve chain specificity sequencing.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol:chloroform:isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μl H2 O. Second-strand cDNA was synthesized by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 4 μl 10 mM dNTPs with dTTP replaced by dUTP (Sigma - Aldrich, St Louis, MO, USA), 30 μl 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 hours. .. Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR.

    Article Title: Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes
    Article Snippet: Second-strand cDNA was synthesized using dNTPs (dUTP replaced dTTP), buffer, RNaseH, and DNA polymerase I. .. The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ].

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: Second-strand cDNA was synthesized with a dNTP mix containing dUTP instead of dTTP. .. After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Picogreen Assay:

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker. .. After Exonuclease I (NEB) treatment, the resulting second-strand cDNA was purified with Agencourt AMPure XP.

    Construct:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs). .. The reactions were incubated at 37 °C for 45 min, followed by heating to 80 °C and slow cooling to 22 °C at 0.1 °C/s in a temperature-controlled block.

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. The assembly mixture was slowly cooled to 12 °C at 0.1 °C/s in a thermocycler to promote annealing of the freshly generated ends of the two USER junctions.

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Approximately 3 μg of RNA per sample were used to construct a complementary DNA (cDNA) library, according to the following procedures: the ribosome RNA (rRNA) was removed and strand-specific RNA-seq libraries were then generated using rRNA-depleted RNA. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: After second-strand cDNA synthesis, sequencing libraries were constructed as described [ ]. .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: RNA-seq libraries were constructed following standard protocols (Illumina TruSeq RNA Sample Prep Kit). .. After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Real-time Polymerase Chain Reaction:

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The oligonucleotides were amplified by PCR in 96 reactions (100 μl each) with 0.02 nM template oligonucleotide, 400 nM each of pAP1V61U primer and AP2V6 primer , and 50 μl of KAPA SYBG fast Universal 2× qPCR Master Mix (Kapabiosystems) at 95 °C for 30 s, 15-16 cycles of 95 °C for 3 s; 55 °C for 30 s; and 60 °C for 20 s, and 60 °C for 2 min. .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Incubation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The resulting single-strand amplicons were purified with Qiaquick PCR purification column. .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min. .. The mixture was digested with 50 units DpnII (10U/μl, NEB) in NEBuffer DpnII at 37 °C for 2 h. Then the mixture was further digested with 5 units USER at 37 °C for 2 h followed by enzyme inactivation at 75 °C for 20 min.

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: After second-strand cDNA synthesis, sequencing libraries were constructed as described [ ]. .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase. .. High-throughput sequencing was performed by UCSD IGM Genomics Center.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: First, five times less adapter mix was ligated to the cDNAs. .. Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs) and 2 M betaine (Sigma).

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: The digested amplicons were purified using ssDNA/RNA clean & concentrator kit (Zymo) using the manufacturer’s protocol. .. Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C. .. Next, 5 µl of 100 µM guide oligo (5′-GTGTATCCTGATC-3′), 2 µl of 10 × DpnII buffer and 8 µl of water were added to the mix and the reaction was incubated at 94 °C for 2 min, followed by 3 min at 37 °C.

    Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
    Article Snippet: First, five times less adapter mixture was ligated to the cDNAs. .. Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs, USA) and 2 M betaine (Sigma, USA).

    Article Title: Sequencing of first-strand cDNA library reveals full-length transcriptomes
    Article Snippet: The ligated samples were size-selected using 1.8 volumes of RNAClean XP beads (Beckman Coulter) with a 40-min incubation. .. The samples were incubated with 2 µl of USER (NEB) at 37 °C for 2 h to degrade the non-ligated strands of the splint adaptors and were then column purified. .. In general, we followed the initial protocol for dUTP library preparation , with minor modifications.

    Stripping Membranes:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. With a library of constructs in hand, we removed denatured enzymes and reaction buffer from the assembly mixture by adding 5 vol of PB buffer (Qiagen) to the assembly reaction mixture and binding the material onto a MinElute column (480 μL per column).

    Expressing:

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: A plasmid encoding the human codon-optimized Streptococcus pyogenes Cas9 nuclease with an NLS and 3×FLAG tag (Addgene plasmid 43861) was used as the wild-type Cas9 expression plasmid. .. 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 . .. Plasmid constructs generated in this work will be deposited with Addgene.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments. .. After amplification of the libraries, samples with unique sample indexes were pooled and sequenced using HiSeq 2000 with TruSeq SBS Kit v3 reagent or HiSeq 2500 with TruSeq SBS Kit v4 reagent (Illumina) following the manufacturer's protocols.

    Genome Wide:

    Article Title: Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX
    Article Snippet: In the following sections, we applied epiPALEOMIX to HTS data generated following three main experimental procedures to explore the presence of genome-wide epigenetic signatures in a wide range of ancient samples, including different tissue types (hair, bones and teeth), geographic origins and DNA preservation conditions. .. In the first experimental procedure, aDNA extracts were treated with USER (New England Biolabs), a commercial DNA repair mix containing Uracil DNA Glycosylase (UNG) and Endonuclease VIII (EndoVIII) (method 1-USER, orange figure header backgrounds).

    Modification:

    Article Title: Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
    Article Snippet: Outer cycling was performed with general handles incorporated from PCR1, but including a uridine modification in the indexed end. .. The modification was used after PCR2 by incubating 1 U USER (NEB) per μg input DNA for 30 minutes at 37°C, thereby protecting the indexed end from exonuclease degradation. .. For robust performance an MBS Magnatrix 1200 (NorDiag) automatic workstation was used to perform the enzymatic steps involved in degradation and sub-sampling of the libraries.

    Transformation Assay:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs). .. The reactions were incubated at 37 °C for 45 min, followed by heating to 80 °C and slow cooling to 22 °C at 0.1 °C/s in a temperature-controlled block.

    Derivative Assay:

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: The derived oxidized dialdehyde of the cap site and 3′ ends of the RNA strands were biotinylated with biotin (long arm) hydrazide (Vector Laboratories) and treated with RNaseONE (Promega) in order to remove the 3′ end of each RNA strand and the biotinylated cap when cDNA failed to reach the 5′ ends. .. The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Second strand was generated using dUTP instead of dTTP to tag the second strand. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments. .. After amplification of the libraries, samples with unique sample indexes were pooled and sequenced using HiSeq 2000 with TruSeq SBS Kit v3 reagent or HiSeq 2500 with TruSeq SBS Kit v4 reagent (Illumina) following the manufacturer's protocols.

    Ligation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Second strand was generated using dUTP instead of dTTP to tag the second strand. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments. .. After amplification of the libraries, samples with unique sample indexes were pooled and sequenced using HiSeq 2000 with TruSeq SBS Kit v3 reagent or HiSeq 2500 with TruSeq SBS Kit v4 reagent (Illumina) following the manufacturer's protocols.

    Article Title: Sequencing of first-strand cDNA library reveals full-length transcriptomes
    Article Snippet: The ligation was initiated by adding 50 µl of ligase mix containing 4 µl of 10 × T4 DNA ligase buffer (NEB), 1 µl of 10 mg/ml BSA (NEB), 2 µl of Quick T4 DNA ligase (NEB) and 33 µl of 2X Quick ligase buffer (NEB). .. The samples were incubated with 2 µl of USER (NEB) at 37 °C for 2 h to degrade the non-ligated strands of the splint adaptors and were then column purified.

    Infection:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Generated:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. Generally, each round of evolution required ~1 mL of USER assembly mixture (22 nmol of each DNA assembly fragment) which was distributed into 10-μL aliquots across multiple 8-well PCR strips.

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Approximately 3 μg of RNA per sample were used to construct a complementary DNA (cDNA) library, according to the following procedures: the ribosome RNA (rRNA) was removed and strand-specific RNA-seq libraries were then generated using rRNA-depleted RNA. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Second strand was generated using dUTP instead of dTTP to tag the second strand. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments.

    Article Title: Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX
    Article Snippet: In the following sections, we applied epiPALEOMIX to HTS data generated following three main experimental procedures to explore the presence of genome-wide epigenetic signatures in a wide range of ancient samples, including different tissue types (hair, bones and teeth), geographic origins and DNA preservation conditions. .. In the first experimental procedure, aDNA extracts were treated with USER (New England Biolabs), a commercial DNA repair mix containing Uracil DNA Glycosylase (UNG) and Endonuclease VIII (EndoVIII) (method 1-USER, orange figure header backgrounds).

    Polymerase Chain Reaction:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: All PCR products were purified using MinElute PCR Purification Kit (Qiagen) to 10 μL final volume and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Each PCR reaction was combined with 300 mL of PB DNA binding buffer (Qiagen) and 25 mL of the solution was loaded onto a HiBind DNA Midi column (Omega Bio-Tek). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The resulting single-strand amplicons were purified with Qiaquick PCR purification column. .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Article Title: Genome-wide profiling reveals cancer-related genes with switched alternative polyadenylation sites in colorectal cancer
    Article Snippet: Next, Illumina p5/p7 adaptors were ligated to the released cDNA. .. Before PCR amplification, the second strand of cDNA which had dUTP was digested by USER (NEB) to achieve chain specificity sequencing. .. Finally, a series of 3T-seq libraries were sequenced by Illumina Hiseq X Ten.

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: After second-strand cDNA synthesis, sequencing libraries were constructed as described [ ]. .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase. .. High-throughput sequencing was performed by UCSD IGM Genomics Center.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: First, five times less adapter mix was ligated to the cDNAs. .. Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs) and 2 M betaine (Sigma).

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: The amplicons were re-purified using QIAquick PCR purification kit (Qiagen) following the manufacturer’s recommendation. .. Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C.

    Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
    Article Snippet: First, five times less adapter mixture was ligated to the cDNAs. .. Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs, USA) and 2 M betaine (Sigma, USA).

    Article Title: Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes
    Article Snippet: The cDNA templates were purified using a Qiagen kit (Venlo, Netherlands) followed by end repair, poly A tailing, and adaptor connection. .. The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ]. .. Finally, the library was sequenced using an IlluminaHiSeq 2000.

    Article Title: Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
    Article Snippet: Paragraph title: PCR and index protection ... The modification was used after PCR2 by incubating 1 U USER (NEB) per μg input DNA for 30 minutes at 37°C, thereby protecting the indexed end from exonuclease degradation.

    Sequencing:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Paragraph title: SMRTbell library preparation and SMRT Sequencing ... Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Paragraph title: 2.4. RNA Isolation, Library Preparation, and Sequencing ... Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Genome-wide profiling reveals cancer-related genes with switched alternative polyadenylation sites in colorectal cancer
    Article Snippet: Next, Illumina p5/p7 adaptors were ligated to the released cDNA. .. Before PCR amplification, the second strand of cDNA which had dUTP was digested by USER (NEB) to achieve chain specificity sequencing. .. Finally, a series of 3T-seq libraries were sequenced by Illumina Hiseq X Ten.

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: After second-strand cDNA synthesis, sequencing libraries were constructed as described [ ]. .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: Paragraph title: CAGE library preparation and sequencing ... The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Second strand was generated using dUTP instead of dTTP to tag the second strand. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments. .. After amplification of the libraries, samples with unique sample indexes were pooled and sequenced using HiSeq 2000 with TruSeq SBS Kit v3 reagent or HiSeq 2500 with TruSeq SBS Kit v4 reagent (Illumina) following the manufacturer's protocols.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR.

    Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
    Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2 O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. .. Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes
    Article Snippet: The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ]. .. The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ].

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: The reaction contained samples eluted in 50 µl resuspension buffer, 2 µl 5× FS buffer, 1 µl 50 mM MgCl2, 1 µl 100 mM DTT, 2 µl 10 mM dUTP nucleotides mix, 15 µl Second Strand Buffer (Invitrogen), 0.5 µl E.coli DNA Ligase (10 U/µl;NEB), 0.5 µl RNase H (2 U/µl;Invitrogen), 2 µl DNA E.coli Polymerase I (10 U/µl;NEB). .. After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA. .. The samples were put in ice and then subjected to PCR amplification.

    Binding Assay:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Each PCR reaction was combined with 300 mL of PB DNA binding buffer (Qiagen) and 25 mL of the solution was loaded onto a HiBind DNA Midi column (Omega Bio-Tek). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Nucleic Acid Electrophoresis:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    RNA Sequencing Assay:

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Approximately 3 μg of RNA per sample were used to construct a complementary DNA (cDNA) library, according to the following procedures: the ribosome RNA (rRNA) was removed and strand-specific RNA-seq libraries were then generated using rRNA-depleted RNA. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: Paragraph title: RNA-seq ... 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Paragraph title: RNA-sequencing (RNA-seq) and data analysis ... Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments.

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: Paragraph title: Validation of exon candidates by RT-PCR and RNA-seq ... After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Paragraph title: Nuclear strand-specific RNA-Seq ... Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads.

    Magnetic Beads:

    Article Title: Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes
    Article Snippet: Poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads for eukaryotic mRNA. .. The samples then were treated with the USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Massachusetts, U.S.A) to digest the antisense strand DNA as insert size of 200 to 300 bases, and then used for PCR amplification [ ].

    Isolation:

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Paragraph title: 2.4. RNA Isolation, Library Preparation, and Sequencing ... Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: In short, mRNA was isolated from 1 μg total RNA using Dynabeads Oligo(dT)25 (Life Technologies) and fragmented to 150–200 nt in first strand buffer for 3 min at 94°C. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments.

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Nuclear RNA was isolated using TRIsure (Bioline), treated with DNaseI (Roche) and re-purified using an RNeasy Mini Kit (Qiagen). .. Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads.

    Functional Assay:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Purification:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: All PCR products were purified using MinElute PCR Purification Kit (Qiagen) to 10 μL final volume and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Following size selection, size-selected cDNA was re-amplified using LongAmp HotStart Taq. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends. .. The the sticky ended cDNA for each size-bin was then ligated to 1 μM hairpin adapters that contain complementary sticky ends to create SMRTbell templates using 2000 Units T4 DNA ligase (New England Biolabs).

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The resulting single-strand amplicons were purified with Qiaquick PCR purification column. .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: Poly(A)+ RNA was enriched with Dynabeads mRNA Purification Kit (Life Technologies, Cat#61006). .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: The cDNA was purified with Agencourt AMPure XP, and then a 3′ linker containing an Illumina adapter sequence was ligated to it. .. The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker. .. Second-strand cDNA was synthesized with a second primer consisting of another Illumina adapter sequence.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs) and 2 M betaine (Sigma).

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: The digested amplicons were purified using ssDNA/RNA clean & concentrator kit (Zymo) using the manufacturer’s protocol. .. Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C.

    Article Title: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
    Article Snippet: Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs, USA) and 2 M betaine (Sigma, USA).

    Article Title: Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
    Article Snippet: After the first PCR the samples were automatically purified using polyethylene glycol precipitation and magnetic bead capture as we described previously . .. The modification was used after PCR2 by incubating 1 U USER (NEB) per μg input DNA for 30 minutes at 37°C, thereby protecting the indexed end from exonuclease degradation.

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Following purification on QIAquick columns (Qiagen), TruSeq Illumina adapters were ligated with T4 DNA Ligase (Enzymatics). .. Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads. .. Libraries were amplified with 9–11 PCR cycles and sequenced (50 bp paired end) on a HiSeq1000 platform (Illumina).

    Article Title: Sequencing of first-strand cDNA library reveals full-length transcriptomes
    Article Snippet: The ligated samples were size-selected using 1.8 volumes of RNAClean XP beads (Beckman Coulter) with a 40-min incubation. .. The samples were incubated with 2 µl of USER (NEB) at 37 °C for 2 h to degrade the non-ligated strands of the splint adaptors and were then column purified. .. In general, we followed the initial protocol for dUTP library preparation , with minor modifications.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: Paragraph title: Validation of exon candidates by RT-PCR and RNA-seq ... After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min. .. The USER/DpnII digested DNAs were purified with Qiaquick PCR purification column.

    cDNA Library Assay:

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: Approximately 3 μg of RNA per sample were used to construct a complementary DNA (cDNA) library, according to the following procedures: the ribosome RNA (rRNA) was removed and strand-specific RNA-seq libraries were then generated using rRNA-depleted RNA. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: Paragraph title: Strand-specific cDNA library ... Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: Poly(A)+ RNA was enriched with Dynabeads mRNA Purification Kit (Life Technologies, Cat61006). .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Polymerase Cycling Assembly:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: All PCR products were purified using MinElute PCR Purification Kit (Qiagen) to 10 μL final volume and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs). .. The reactions were incubated at 37 °C for 45 min, followed by heating to 80 °C and slow cooling to 22 °C at 0.1 °C/s in a temperature-controlled block.

    Plasmid Preparation:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Both DNA fragments were quantified using a NanoDrop 1000 Spectrophotometer (Themo Fisher Scientific). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs). .. Generally, each round of evolution required ~1 mL of USER assembly mixture (22 nmol of each DNA assembly fragment) which was distributed into 10-μL aliquots across multiple 8-well PCR strips.

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity
    Article Snippet: A plasmid encoding the human codon-optimized Streptococcus pyogenes Cas9 nuclease with an NLS and 3×FLAG tag (Addgene plasmid 43861) was used as the wild-type Cas9 expression plasmid. .. 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 . .. Plasmid constructs generated in this work will be deposited with Addgene.

    Software:

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker. .. The cDNA concentration of the final product was determined with a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).

    Multiplex Assay:

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Next, the backbone of the bacterial base editor plasmid template , was amplified with primers NMG-799 and NMG-824 ( ) and Phusion U Green Multiplex PCR Master Mix (100 μL per well in a 98-well PCR plate, 5–6 plates total, Tm 66 °C, 4.5-min extension) following the manufacturer’s protocol. .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Selection:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Following size selection, size-selected cDNA was re-amplified using LongAmp HotStart Taq. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: After first strand cDNA synthesis, remaining dNTPs were removed by a size selection on beads (AMPure XP). .. After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Sample Prep:

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
    Article Snippet: RNA-seq libraries were constructed following standard protocols (Illumina TruSeq RNA Sample Prep Kit). .. After sequencing adaptors were ligated, 1 µl USER (Uracil-Specific Excision Reagent enzyme; NEB) was added to reactions to degrade the second strand cDNA.

    Size-exclusion Chromatography:

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: 250 ng nuclear RNA was fragmented using a Covaris E220 instrument at standard RNA settings for 60 sec. Fragmented RNA was precipitated and first strand synthesis was carried out using SuperScript III (Invitrogen) with 4 µg of actinomycin D (Sigma). .. Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads.

    Ethanol Precipitation:

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: The amplified amplicons were purified by ethanol precipitation and re-purified with Qiaquick PCR purification columns (Qiagen). .. Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min.

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: First-strand cDNA was cleaned up by extraction twice with phenol:chloroform:isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μl H2 O. Second-strand cDNA was synthesized by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 4 μl 10 mM dNTPs with dTTP replaced by dUTP (Sigma - Aldrich, St Louis, MO, USA), 30 μl 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 hours. .. Second, 1 U USER (New England Biolabs - Ipswich, MA, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C before PCR.

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: The amplicons were then pooled and concentrated using ethanol precipitation. .. Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C.

    Preserving:

    Article Title: Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX
    Article Snippet: In the following sections, we applied epiPALEOMIX to HTS data generated following three main experimental procedures to explore the presence of genome-wide epigenetic signatures in a wide range of ancient samples, including different tissue types (hair, bones and teeth), geographic origins and DNA preservation conditions. .. In the first experimental procedure, aDNA extracts were treated with USER (New England Biolabs), a commercial DNA repair mix containing Uracil DNA Glycosylase (UNG) and Endonuclease VIII (EndoVIII) (method 1-USER, orange figure header backgrounds).

    Random Hexamer Labeling:

    Article Title: Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function
    Article Snippet: For each library, 500 ng poly(A)+ RNA was premixed with 0.2 μg of Oligo(dT) and random hexamer primer, heated to 70°C for 10 min., and chilled on ice. .. 250 to 500 bp size-selected, adaptor-ligated cDNA was incubated with 1 U USER (NEB) at 37°C for 15 min. followed by 5 min. at 95°C before PCR with Phusion High-Fidelity DNA polymerase.

    Article Title: JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion
    Article Snippet: Random hexamer primed first strand was generated in presence of dATP, dGTP, dCTP and dTTP. .. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments.

    Spectrophotometry:

    Article Title: Continuous evolution of B. thuringiensis toxins overcomes insect resistance
    Article Snippet: All PCR products were purified using MinElute PCR Purification Kit (Qiagen) to 10 μL final volume and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). .. For assembly, PCR products carrying complementary USER junctions were mixed in an equimolar ratio (up to 1 pmol each) in a 10 μL reaction containing 15 units Dpn I (New England Biolabs), 0.75 units USER (Uracil-Specific Excision Reagent) enzyme (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA at pH 7.9 (1x CutSmart Buffer, New England Biolabs).

    Article Title: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
    Article Snippet: Both DNA fragments were quantified using a NanoDrop 1000 Spectrophotometer (Themo Fisher Scientific). .. TadA* libraries were assembled following a previously reported USER assembly procedure with the following conditions: 0.22 pmol of ecTadA mutagenized DNA fragment 1, 0.22 pmol of plasmid backbone fragment 2, 1 U of USER (Uracil-Specific Excision Reagent, New England Biolabs) enzyme, and 1 U of DpnI enzyme (New England Biolabs) per 10 μL of USER assembly mixture were combined in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/mL BSA at pH7.9 (1× CutSmart Buffer, New England Biolabs).

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: The purity, concentration, and integrity of the RNA were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Evaporation:

    Article Title: Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging
    Article Snippet: Second, 3–5 µg of the single-stranded probes were then digested with 5 units of USER (New England Biolabs) in an 80 µl reaction containing 1 × DpnII buffer (New England Biolabs) and incubated for 1 h at 37 °C. .. The probes were then purified using a TBE-Urea denaturing gel and cutting the band corresponding to ~ 120 bp.

    Concentration Assay:

    Article Title: Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis
    Article Snippet: The purity, concentration, and integrity of the RNA were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. .. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK).

    Article Title: Retinal transcriptome profiling at transcription start sites: a cap analysis of gene expression early after axonal injury
    Article Snippet: The cDNA was again purified with Agencourt AMPure XP, followed by treatment with Shrimp Alkaline Phosphatase (Affymetrics) and USER (NEB) to restrict the upper strand of the 3′ linker. .. After Exonuclease I (NEB) treatment, the resulting second-strand cDNA was purified with Agencourt AMPure XP.

    Fractionation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends.

    Two-Dimensional Gel Electrophoresis:

    Article Title: Library-free Methylation Sequencing with Bisulfite Padlock Probes
    Article Snippet: Approximately 5-8 μg of single strand amplicons were subsequently digested with 5 units USER (1 U/μl, NEB) at 37 °C for 1 h. The digested DNAs were annealed to 5.88 μM RE-DpnII-V6 guide oligo ( ) and denatured at 94 °C for 2 min decreased the temperature to 37 °C and incubated at 37 °C for 3 min. .. The USER/DpnII digested DNAs were purified with Qiaquick PCR purification column.

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    New England Biolabs user enzyme mix
    Primer sequences for amplifying the different <t>USER-Bricks,</t> specifying template, product size and compatible USER-Brick fragments. PCR templates : A = pAg1-H3, pRF-HU2 [ 15 ], B = pPK2 or pPZP-201BK [ 16 ], C = pANT-hyg(R) [ 17 ] or pCSN43 (Fungal Genetics Stock Center), D = pAN7-1 [ 18 ], E = pSM334 [ 19 ], F = pBARKS1 [ 20 ], G = A. nidulans genomic <t>DNA</t> or pRF-HUE, pRF-HU2E [ 15 ], H = pWJ1350 [ 21 ] or plasmids derived from the original Discosoma sp. study [ 22 ]. In primer sequences: U = 2-deoxyuridine.
    User Enzyme Mix, 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
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    Primer sequences for amplifying the different USER-Bricks, specifying template, product size and compatible USER-Brick fragments. PCR templates : A = pAg1-H3, pRF-HU2 [ 15 ], B = pPK2 or pPZP-201BK [ 16 ], C = pANT-hyg(R) [ 17 ] or pCSN43 (Fungal Genetics Stock Center), D = pAN7-1 [ 18 ], E = pSM334 [ 19 ], F = pBARKS1 [ 20 ], G = A. nidulans genomic DNA or pRF-HUE, pRF-HU2E [ 15 ], H = pWJ1350 [ 21 ] or plasmids derived from the original Discosoma sp. study [ 22 ]. In primer sequences: U = 2-deoxyuridine.

    Journal: BMC Molecular Biology

    Article Title: Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    doi: 10.1186/1471-2199-15-15

    Figure Lengend Snippet: Primer sequences for amplifying the different USER-Bricks, specifying template, product size and compatible USER-Brick fragments. PCR templates : A = pAg1-H3, pRF-HU2 [ 15 ], B = pPK2 or pPZP-201BK [ 16 ], C = pANT-hyg(R) [ 17 ] or pCSN43 (Fungal Genetics Stock Center), D = pAN7-1 [ 18 ], E = pSM334 [ 19 ], F = pBARKS1 [ 20 ], G = A. nidulans genomic DNA or pRF-HUE, pRF-HU2E [ 15 ], H = pWJ1350 [ 21 ] or plasmids derived from the original Discosoma sp. study [ 22 ]. In primer sequences: U = 2-deoxyuridine.

    Article Snippet: For USER cloning reactions 1 μl of the needed purified USER-Bricks and 2 μl of the required gene specific inserts were mixed with 1.2 units of ‘USER enzyme mix’ (New England Biolabs) and 10xTaq DNA polymerase buffer (Sigma-Aldrich) to a final concentration of 1x, in a total volume of 12 μl.

    Techniques: Polymerase Chain Reaction, Derivative Assay

    Design of vectors for random heterologous expression with the gene’s natural promoter (A), with an alternative promoter (B), for targeted gene replacement (C) and in locus overexpression (D). A) Expression of the gene of interest from a random locus in the genome, driven by the gene’s natural promoter. Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. B) Overexpression of the gene of interest from a random genomic locus, with the expression driven by a heterologous promoter, in this case the gpdA promoter from Aspergillus nidulans . Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. C) Replacement of the gene of interest. Note that the HRS1 fragment can also be reused for in locus overexpression experiments. D) In locus overexpression of the gene of interest by targeted integration of a strong constitutive promoter. Note that the HRS1 fragment can be reused for deletion experiments. Primers are represented by solid black arrows. Aberrations: gDNA = genomic DNA; P = promoter; CDS = coding sequence; T = terminator; RB LB = right left borders defining the T-DNA region; T-DNA = transfer DNA.

    Journal: BMC Molecular Biology

    Article Title: Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    doi: 10.1186/1471-2199-15-15

    Figure Lengend Snippet: Design of vectors for random heterologous expression with the gene’s natural promoter (A), with an alternative promoter (B), for targeted gene replacement (C) and in locus overexpression (D). A) Expression of the gene of interest from a random locus in the genome, driven by the gene’s natural promoter. Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. B) Overexpression of the gene of interest from a random genomic locus, with the expression driven by a heterologous promoter, in this case the gpdA promoter from Aspergillus nidulans . Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. C) Replacement of the gene of interest. Note that the HRS1 fragment can also be reused for in locus overexpression experiments. D) In locus overexpression of the gene of interest by targeted integration of a strong constitutive promoter. Note that the HRS1 fragment can be reused for deletion experiments. Primers are represented by solid black arrows. Aberrations: gDNA = genomic DNA; P = promoter; CDS = coding sequence; T = terminator; RB LB = right left borders defining the T-DNA region; T-DNA = transfer DNA.

    Article Snippet: For USER cloning reactions 1 μl of the needed purified USER-Bricks and 2 μl of the required gene specific inserts were mixed with 1.2 units of ‘USER enzyme mix’ (New England Biolabs) and 10xTaq DNA polymerase buffer (Sigma-Aldrich) to a final concentration of 1x, in a total volume of 12 μl.

    Techniques: Expressing, Over Expression, Selection, Marker, Plasmid Preparation, Sequencing

    The different DNA fragments (=bricks) in the USER-Brick vector system. The ends of the Bricks are colour coded based on which overhangs that are compatible for fusion. Top panel : The core USER-Brick includes backbones, selection markers, promoters and fluorescent marker fragments. Centre panel : The placement of the different types of PCR amplicons in relation to the gene of interest. Bottom panel : Sequences of the 5′ overhang found on the primers for amplifying the different USER-Bricks in the two panels above.

    Journal: BMC Molecular Biology

    Article Title: Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    doi: 10.1186/1471-2199-15-15

    Figure Lengend Snippet: The different DNA fragments (=bricks) in the USER-Brick vector system. The ends of the Bricks are colour coded based on which overhangs that are compatible for fusion. Top panel : The core USER-Brick includes backbones, selection markers, promoters and fluorescent marker fragments. Centre panel : The placement of the different types of PCR amplicons in relation to the gene of interest. Bottom panel : Sequences of the 5′ overhang found on the primers for amplifying the different USER-Bricks in the two panels above.

    Article Snippet: For USER cloning reactions 1 μl of the needed purified USER-Bricks and 2 μl of the required gene specific inserts were mixed with 1.2 units of ‘USER enzyme mix’ (New England Biolabs) and 10xTaq DNA polymerase buffer (Sigma-Aldrich) to a final concentration of 1x, in a total volume of 12 μl.

    Techniques: Plasmid Preparation, Selection, Marker, Polymerase Chain Reaction

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Article Snippet: Subsequently, a mixture of PCR product, 1 U USER™ enzyme mix (New England Biolabs), and PacI/Nt.BbvCI digested USER vector was incubated 20 min at 37°C followed by 20 min at 25°C and finally transformed into chemically competent Escherichia coli cells (do not use electroshock transformation).

    Techniques: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Article Snippet: Subsequently, a mixture of PCR product, 1 U USER™ enzyme mix (New England Biolabs), and PacI/Nt.BbvCI digested USER vector was incubated 20 min at 37°C followed by 20 min at 25°C and finally transformed into chemically competent Escherichia coli cells (do not use electroshock transformation).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    Overview of pUCE vectors . A USER TC-TG site was introduced in the plasmid pWBVec8 to generate pUCE as described in the text. pUCE D-Hord:HvHB1:NOS was generated from pUCE in a single step by combining PCR products of D-Hordein promoter, HvHB1 cDNA and NOS. pUCE UBI:GFP-ATG8:NOS was generated from pUCE UBI:USER:NOS in a single step by combining PCR products of eGFP and ATG8. The remaining vectors were generated by insertion of single PCR products. A USER™ site is lost when a PCR product is inserted. However, it can be reconstituted if included in one of the primers of the PCR reaction.

    Journal: Plant Methods

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals

    doi: 10.1186/1746-4811-6-15

    Figure Lengend Snippet: Overview of pUCE vectors . A USER TC-TG site was introduced in the plasmid pWBVec8 to generate pUCE as described in the text. pUCE D-Hord:HvHB1:NOS was generated from pUCE in a single step by combining PCR products of D-Hordein promoter, HvHB1 cDNA and NOS. pUCE UBI:GFP-ATG8:NOS was generated from pUCE UBI:USER:NOS in a single step by combining PCR products of eGFP and ATG8. The remaining vectors were generated by insertion of single PCR products. A USER™ site is lost when a PCR product is inserted. However, it can be reconstituted if included in one of the primers of the PCR reaction.

    Article Snippet: USER™ cloning was performed in a total volume of 12 μl (10 μl PCR product, 1 μl USER™ Enzyme mix (New England Biolabs), 1 μl pre-digested USER Vector 50-100 ng/μl).

    Techniques: Plasmid Preparation, Generated, Polymerase Chain Reaction

    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Journal: Plant Methods

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals

    doi: 10.1186/1746-4811-6-15

    Figure Lengend Snippet: Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Article Snippet: USER™ cloning was performed in a total volume of 12 μl (10 μl PCR product, 1 μl USER™ Enzyme mix (New England Biolabs), 1 μl pre-digested USER Vector 50-100 ng/μl).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    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