user enzyme mix  (New England Biolabs)


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  • 99
    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 enzyme mix
    USER Enzyme
    USER Enzyme 250 units
    https://www.bioz.com/result/user enzyme mix/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    user enzyme mix - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system"

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

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-15

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    2) Product Images from "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl635

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    3) Product Images from "Simultaneous and stoichiometric purification of hundreds of oligonucleotides"

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04870-w

    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
    Figure Legend 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

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

    4) Product Images from "UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals"

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

    Journal: Plant Methods

    doi: 10.1186/1746-4811-6-15

    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.
    Figure Legend 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.

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    Related Articles

    Clone Assay:

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: The remaining hα1AT and hC1INH constructs ( ) were cloned by seamless PCR-based uracil-specific excision reagent (USER) fusion cloning, essentially as previously described , using primers designed by the AMUSER software . .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals
    Article Snippet: The set of oligonucleotides and primers used to generate DNA fragments for cloning is described in Additional file , Table S2 for GBSS Transit Peptide and Additional file , Table S3 for D-hordein signal peptide. .. 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). .. The USER™ Enzyme mix is fully functional in the PCR buffer and the reaction can be performed directly with the unpurified PCR product reaction.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs). .. The resulting vector was pCfB322, which has already been used in a metabolic study by Borodina et al. [ ]. pCfB322 bears an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker fused to the degradation signal (Kl .URA3-degradation signal ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family.

    Article Title: Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency
    Article Snippet: RNA was isolated from primary CD56+ NK cells and CD8+ T cells from the peripheral blood of healthy donors, and 5′ RACE was performed with the 5′/3′ RACE kit, second generation (Roche) using primers listed in Table S1 . .. pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607). .. A version of the intron 1 promoter with the c.118-308C > T mutation and a full-length version of the UNC13D promoter (−1,374 to +607) were also created by USER cloning.

    Amplification:

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: Solid-phase cloning (SPC) was used to fuse the S11_M3-tag to the C-terminus of hC1INH. pcDNA3.1(+)/Zeo encoding hC1INH (PL_hC1INH_notag; ) was PCR amplified using Phusion® Hot-Start Flex (New England Biolabs, Ipswich, Massachusetts, USA) and primers #131 and #132. .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. .. After the incubation, 1 μl of T4 DNA Polymerase (Thermo Fisher Scientific) was added to the mix, and the mix was further incubated at 25°C for 15 min and at 12°C for 5 min.

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: Libraries for Illumina sequencing [ ] were made with NEB reagent kits and paired-end adapters using modified PCR amplification conditions to minimize base-composition bias [ ]. .. Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs). .. As reporter, a GFP expression cassette comprising the S . cerevisiae TEF1 promoter and the coding sequence for the green fluorescent protein was cloned using the classical uracil excision protocol reported previously [ ].

    Synthesized:

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: First strand cDNA was reverse transcribed using random hexamer primers, with second strand cDNA synthesized subsequently. .. Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H− ). .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH− ). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (NaseH− ). .. Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: First strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H−). .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: First-strand cDNA was synthesized using random hexamer primer. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNaseH− ). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Construct:

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: The remaining hα1AT and hC1INH constructs ( ) were cloned by seamless PCR-based uracil-specific excision reagent (USER) fusion cloning, essentially as previously described , using primers designed by the AMUSER software . .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: To assess the transcriptomes of the clams and obtain a quantitative and qualitative gene expression database for low salinity stress, four R. philippinarum cDNA libraries representing the control group (SRp1, SRp2) and the treated group (FRp1, FRp2) were constructed. .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Twelve cDNA libraries (three replicates for each sample) were constructed using the NEBNext® Ultra™RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's protocol. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency
    Article Snippet: RNA was isolated from primary CD56+ NK cells and CD8+ T cells from the peripheral blood of healthy donors, and 5′ RACE was performed with the 5′/3′ RACE kit, second generation (Roche) using primers listed in Table S1 . .. pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607). .. A version of the intron 1 promoter with the c.118-308C > T mutation and a full-length version of the UNC13D promoter (−1,374 to +607) were also created by USER cloning.

    Real-time Polymerase Chain Reaction:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. .. After the incubation, 1 μl of T4 DNA Polymerase (Thermo Fisher Scientific) was added to the mix, and the mix was further incubated at 25°C for 15 min and at 12°C for 5 min.

    Random Hexamer Labeling:

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: First strand cDNA was reverse transcribed using random hexamer primers, with second strand cDNA synthesized subsequently. .. Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H− ). .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH− ). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: Next, a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H- ) were used to synthesize the first strand cDNA. .. Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (NaseH− ). .. Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: Random hexamer primer and M-MuLV Reverse Transcriptase (RNase H) was used to synthesize the first strand cDNA. .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: First strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H−). .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: First-strand cDNA was synthesized using random hexamer primer. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNaseH− ). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Luciferase:

    Article Title: Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency
    Article Snippet: RNA was isolated from primary CD56+ NK cells and CD8+ T cells from the peripheral blood of healthy donors, and 5′ RACE was performed with the 5′/3′ RACE kit, second generation (Roche) using primers listed in Table S1 . .. pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607). .. A version of the intron 1 promoter with the c.118-308C > T mutation and a full-length version of the UNC13D promoter (−1,374 to +607) were also created by USER cloning.

    Activity Assay:

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: Subsequently, second-strand cDNA synthesis was performed using second-strand synthesis reaction buffer, DNA polymerase I, and RNase H. Remaining overhangs were converted into blunt ends by exonuclease/polymerase activity. .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency
    Article Snippet: pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607). .. pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607).

    Expressing:

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: Paragraph title: Construction of cDNA libraries for digital gene expression sequencing ... Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: To assess the transcriptomes of the clams and obtain a quantitative and qualitative gene expression database for low salinity stress, four R. philippinarum cDNA libraries representing the control group (SRp1, SRp2) and the treated group (FRp1, FRp2) were constructed. .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs). .. The resulting vector was pCfB322, which has already been used in a metabolic study by Borodina et al. [ ]. pCfB322 bears an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker fused to the degradation signal (Kl .URA3-degradation signal ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family.

    Modification:

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: Libraries for Illumina sequencing [ ] were made with NEB reagent kits and paired-end adapters using modified PCR amplification conditions to minimize base-composition bias [ ]. .. Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Kl .URA3 on pCfB312 was further modified by the addition of a degradation signal for quicker degradation of the Ura3 protein [ ]. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

    Gel Purification:

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: This was done by amplifying pCfB312 using primers PR-521 and PR-522 using PfuX7 [ ]. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs). .. The resulting vector was pCfB322, which has already been used in a metabolic study by Borodina et al. [ ]. pCfB322 bears an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker fused to the degradation signal (Kl .URA3-degradation signal ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family.

    Ligation:

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: To simplify and streamline the process, especially for low input libraries, we transitioned to the 1 tube 'with bead' method [ ] in which all the steps (end repair, A-base addition and adaptor ligation ± indexing) were carried out in a single tube. .. Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes. .. Samples were enriched with Illumina PE1.0 and PE2.0 primers (1 μM each), 1× of AccuPrime PCR buffer I (10×), 0.5 U of AccuPrime Taq High Fidelity polymerase (5 U/μl; Invitrogen) in a final volume of 25 μl.

    Generated:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Sequencing libraries of six samples (HP group, n =3; LP group, n =3) were generated using NEBNextR Ultra™ Directional RNA Library Prep Kit for IlluminaR (NEB, U.S.A.) following the manufacturer’s recommendations, and index codes were used to label the sequences of each sample. .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR. .. Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: The sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), according to the manufacturer’s recommendations, and index codes were added to attribute the sequences to each sample. .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments. .. PCR products were purified with the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system.

    Polymerase Chain Reaction:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: To select fragments of 150–200 bp, the library fragments were purified with AMPure XP Beads (Beckman Coulter, Beverly, U.S.A.). .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: In order to select cDNA fragments of preferentially 150 ~ 200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: The remaining hα1AT and hC1INH constructs ( ) were cloned by seamless PCR-based uracil-specific excision reagent (USER) fusion cloning, essentially as previously described , using primers designed by the AMUSER software . .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL). .. All target genes were cloned into a vector harbouring an ‘Nt.BbvCI/PacI A’ USER cassette (PL_TGExpr) ( ).

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: In order to select cDNA fragments with a preference of 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, USA). .. Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: In order to select cDNA fragments of preferentially 150–200 bp length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: To select cDNA fragments that are 150–200 bp in length, the fragments in each of the library were purified with an AMPure XP system (Beckman Coulter, Brea, CA, USA). .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. The qPCRs were performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: In order to select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR. .. Then PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: To select cDNA fragments of 150–200 bp in length, the AMPure XP system (Beckman Coulter, Beverly, USA) was used. .. Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR. .. PCR was then performed with the Phusion High-Fidelity DNA polymerase, Universal PCR primers and the Index (X) Primer.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: To select cDNA fragments of preferential 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: NEBNext Adaptor were ligated to prepare for hybridization after adenylation of 3′ ends of DNA fragments. .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. .. PCR products were purified by AMPure XP system and library quality was assessed using Agilent Bioanalyzer 2100 system.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: To preferentially select cDNA fragments of 150–200 bp, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR. .. The PCR was performed with Phusion High-Fidelity DNA Polymerase, universal PCR primers, and Index (X) Primer.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: After adenylation of 3′ ends of DNA fragments, a NEB Next Adaptor with hairpin loop structure was ligated to prepare for hybridization. .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. PCR was performed for 10 cycles with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments. .. Then PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index(X) Primer.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: To preferentially select 150–200 bp cDNA fragments, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. The PCR assay was then performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and an Indexed (X) Primer.

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. .. After the incubation, 1 μl of T4 DNA Polymerase (Thermo Fisher Scientific) was added to the mix, and the mix was further incubated at 25°C for 15 min and at 12°C for 5 min.

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: Libraries for Illumina sequencing [ ] were made with NEB reagent kits and paired-end adapters using modified PCR amplification conditions to minimize base-composition bias [ ]. .. Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes.

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals
    Article Snippet: The set of oligonucleotides and primers used to generate DNA fragments for cloning is described in Additional file , Table S2 for GBSS Transit Peptide and Additional file , Table S3 for D-hordein signal peptide. .. 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). .. The USER™ Enzyme mix is fully functional in the PCR buffer and the reaction can be performed directly with the unpurified PCR product reaction.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Like above, the two latter PCR fragments share homologous regions at their 5’ and 3’ ends so that In-Fusion reaction can be performed. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

    Article Title: Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency
    Article Snippet: RNA was isolated from primary CD56+ NK cells and CD8+ T cells from the peripheral blood of healthy donors, and 5′ RACE was performed with the 5′/3′ RACE kit, second generation (Roche) using primers listed in Table S1 . .. pGL3 (Promega) luciferase reporter constructs were created by PCR using the USER cloning system (NEB) for the UNC13D intron 1 promoter region (+5 to +607). .. A version of the intron 1 promoter with the c.118-308C > T mutation and a full-length version of the UNC13D promoter (−1,374 to +607) were also created by USER cloning.

    Hybridization:

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: Primer #133 was designed to include 30 bases for hybridization, an amino acid linker (GGGSGGGS), and the S11_M3 sequence. .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. .. Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: After adenylation of the 3’ ends of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to the fragments to prepare them for hybridization. .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated for hybridization. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: After adenylation of the 3’ ends of the DNA fragments, the NEBNext Adaptor with hairpin loop structures was ligated to prepare for hybridization. .. Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. .. Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: NEBNext Adaptor were ligated to prepare for hybridization after adenylation of 3′ ends of DNA fragments. .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: After adenylation of 3′ ends of DNA fragments, a NEB Next Adaptor with hairpin loop structure was ligated to prepare for hybridization. .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: After adenylation of the 3′ ends of cDNA fragments, NEBNext adapter oligonucleotides were ligated to prepare for hybridization, and then the cDNA fragments were purified using AMPure XP system (Beckman Coulter, Beverly, USA) to select the fragments of preferentially 150–200 bp in length. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: After adenylation of the 3′ ends of DNA fragments, NEBNext Adaptors with hairpin loop structures were ligated to prepare for hybridization. .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    RNA Sequencing Assay:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Paragraph title: RNA extraction, library construction, and RNA-seq ... Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: Paragraph title: RNA sequencing and transcriptome data analysis ... Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: Paragraph title: 5.5.2. RNA-Seq Library Construction and Sequencing ... The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: For RNA-Seq, a total amount of 10 μg of RNA per sample was used as material for cDNA library preparations. .. Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Magnetic Beads:

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Life Technologies, Carlsbad, CA, USA), and then fragmented using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: RNAs were subjected to enrichment of poly(A)-tailed mRNAs with poly(T) oligo-attached magnetic beads (Thermo Fisher Scientific, MA, USA) and then fragmented into small pieces using divalent cations at a high temperature. .. Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: Briefly, polyA RNA mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Briefly, mRNA was purified from 1.5 μg of total RNA using oligo (dT) magnetic beads, and then broken into short fragments of 300–500 bp by adding fragmentation buffer. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. .. The libraries were tested using qPCR (as above) and subsequently amplified in five separate reactions of 25 μl with 1 μl of each 10 μM PCR primer (index primer P7 and IS4), 2.5 μl of an AccuPrime Pfx buffer, 0.5 μl of Herculase II Fusion DNA Polymerase (Agilent Technologies), 16 to 17 μl of ddH2 O, and 4 μl of repaired DNA library.

    Isolation:

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: Paragraph title: RNA isolation, library preparation, and sequencing ... To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Purification:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: To select fragments of 150–200 bp, the library fragments were purified with AMPure XP Beads (Beckman Coulter, Beverly, U.S.A.). .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: In order to select cDNA fragments of preferentially 150 ~ 200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: In order to select cDNA fragments with a preference of 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, USA). .. Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: In order to select cDNA fragments of preferentially 150–200 bp length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: To select cDNA fragments that are 150–200 bp in length, the fragments in each of the library were purified with an AMPure XP system (Beckman Coulter, Brea, CA, USA). .. Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: In order to select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR. .. PCR was then performed with the Phusion High-Fidelity DNA polymerase, Universal PCR primers and the Index (X) Primer.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: To select cDNA fragments of preferential 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: NEBNext Adaptor were ligated to prepare for hybridization after adenylation of 3′ ends of DNA fragments. .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. .. PCR products were purified by AMPure XP system and library quality was assessed using Agilent Bioanalyzer 2100 system.

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: To preferentially select cDNA fragments of approximately 200–400 bp, all fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: To preferentially select cDNA fragments of 150–200 bp, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: After adenylation of 3′ ends of DNA fragments, a NEB Next Adaptor with hairpin loop structure was ligated to prepare for hybridization. .. To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. .. PCR was performed for 10 cycles with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: After adenylation of the 3′ ends of cDNA fragments, NEBNext adapter oligonucleotides were ligated to prepare for hybridization, and then the cDNA fragments were purified using AMPure XP system (Beckman Coulter, Beverly, USA) to select the fragments of preferentially 150–200 bp in length. .. Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: To preferentially select 150–200 bp cDNA fragments, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: To simplify and streamline the process, especially for low input libraries, we transitioned to the 1 tube 'with bead' method [ ] in which all the steps (end repair, A-base addition and adaptor ligation ± indexing) were carried out in a single tube. .. Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes. .. Samples were enriched with Illumina PE1.0 and PE2.0 primers (1 μM each), 1× of AccuPrime PCR buffer I (10×), 0.5 U of AccuPrime Taq High Fidelity polymerase (5 U/μl; Invitrogen) in a final volume of 25 μl.

    Sequencing:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Sequencing libraries of six samples (HP group, n =3; LP group, n =3) were generated using NEBNextR Ultra™ Directional RNA Library Prep Kit for IlluminaR (NEB, U.S.A.) following the manufacturer’s recommendations, and index codes were used to label the sequences of each sample. .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms
    Article Snippet: Paragraph title: Library preparation, clustering and sequencing ... Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: Primer #133 was designed to include 30 bases for hybridization, an amino acid linker (GGGSGGGS), and the S11_M3 sequence. .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Article Title: De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance
    Article Snippet: Paragraph title: Library preparation and sequencing ... Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: Paragraph title: RNA extraction, library construction, and sequencing ... Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: Paragraph title: cDNA library construction and transcriptome sequencing ... Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: Paragraph title: RNA extraction, library construction, and sequencing ... Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: Paragraph title: 5.5.2. RNA-Seq Library Construction and Sequencing ... The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: Paragraph title: Construction of cDNA libraries for digital gene expression sequencing ... Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: Paragraph title: Library preparation for Transcriptome sequencing ... The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Article Title: Deep RNA sequencing of pectoralis muscle transcriptomes during late-term embryonic to neonatal development in indigenous Chinese duck breeds
    Article Snippet: Paragraph title: RNA isolation, library preparation, and sequencing ... To select cDNA fragments of 150–200 bp in length, library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA), followed by 3 μl USER Enzyme (NEB) with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Paragraph title: cDNA library construction and sequencing ... Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: Paragraph title: cDNA library construction and Illumina deep sequencing ... Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
    Article Snippet: Paragraph title: Illumina sequencing libraries ... Following adaptor ligation, the purified products were size selected on a gel (approximately 300 to 450 bp). cDNAs created with the second strand dUTP approach were treated with 1 U Uracil-Specific Excision Reagent enzyme mix (USER; NEB) at 37°C for 15 minutes followed by 95°C heat inactivation for 5 minutes.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs). .. The resulting vector was pCfB322, which has already been used in a metabolic study by Borodina et al. [ ]. pCfB322 bears an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker fused to the degradation signal (Kl .URA3-degradation signal ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family.

    Quantitative RT-PCR:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    cDNA Library Assay:

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii)
    Article Snippet: Paragraph title: cDNA library construction and transcriptome sequencing ... Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37 °C for 15 min followed by 95 °C for 5 min before PCR.

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: The fragmented RNAs were reverse-transcribed to produce the final cDNA library using mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s procedure. .. Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium
    Article Snippet: Paragraph title: cDNA library construction and sequencing ... Then 3 μL of USER Enzyme (NEB, USA) was used with size-selected and adaptor-ligated cDNA fragments.

    Article Title: Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour
    Article Snippet: Paragraph title: cDNA library construction and Illumina deep sequencing ... Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Plasmid Preparation:

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals
    Article Snippet: The set of oligonucleotides and primers used to generate DNA fragments for cloning is described in Additional file , Table S2 for GBSS Transit Peptide and Additional file , Table S3 for D-hordein signal peptide. .. 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). .. The USER™ Enzyme mix is fully functional in the PCR buffer and the reaction can be performed directly with the unpurified PCR product reaction.

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Paragraph title: Construction of the EasyCloneMulti vector based on Ty4 cons ... After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

    Software:

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: The remaining hα1AT and hC1INH constructs ( ) were cloned by seamless PCR-based uracil-specific excision reagent (USER) fusion cloning, essentially as previously described , using primers designed by the AMUSER software . .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    RNA Extraction:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: Paragraph title: RNA extraction, library construction, and RNA-seq ... Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: Genome-wide identification and characterization of long non-coding RNAs in developmental skeletal muscle of fetal goat
    Article Snippet: Paragraph title: RNA extraction, library construction, and sequencing ... Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.)
    Article Snippet: Paragraph title: RNA extraction, library construction, and sequencing ... Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Selection:

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Correct vectors corresponding to vector pCfB312, bearing an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker (Kl .URA3 ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family, were verified by restriction digest. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

    Sample Prep:

    Article Title: Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq
    Article Snippet: The fragmented RNAs were reverse-transcribed to produce the final cDNA library using mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s procedure. .. Then, USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min of 95 °C treatment.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: A total amount of 3 μg of RNA per sample was used as the input material for RNA sample preparation. .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR.

    Preserving:

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: On the basis of screening results of conventional blunt-end libraries, one individual from Kazburun 1 (kzb002) was identified as having exceptional preservation. .. Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase.

    Incubation:

    Article Title: Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
    Article Snippet: In order to select cDNA fragments of preferentially 150–200 bp length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected, adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer.

    Article Title: RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum
    Article Snippet: To select cDNA fragments of 150–200 bp in length, the AMPure XP system (Beckman Coulter, Beverly, USA) was used. .. Next, 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR. .. PCR was then performed with the Phusion High-Fidelity DNA polymerase, Universal PCR primers and the Index (X) Primer.

    Article Title: Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
    Article Snippet: NEBNext Adaptor were ligated to prepare for hybridization after adenylation of 3′ ends of DNA fragments. .. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) for selecting cDNA fragments of preferentially 150~200 bp in length. cDNA (size-selected and adaptor-ligated) was incubated with 3 μL USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C, 15 min, followed by at 95 °C for 5 min. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. .. PCR products were purified by AMPure XP system and library quality was assessed using Agilent Bioanalyzer 2100 system.

    Article Title: High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response
    Article Snippet: To preferentially select cDNA fragments of 150–200 bp, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). .. The size-selected adaptor-ligated cDNA was incubated with 3 μl of USER Enzyme (NEB, USA) at 37 °C for 15 min, and then at 95 °C for 5 min before PCR. .. The PCR was performed with Phusion High-Fidelity DNA Polymerase, universal PCR primers, and Index (X) Primer.

    Article Title: Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads
    Article Snippet: To use the individual in a future high-coverage genome database, we commenced production of libraries with repaired damages using uracil-DNA-glycosylase (UDG) treatment to remove deaminated cytosine sites ( ). .. Two new libraries were prepared using the standard blunt-end protocol listed above ( ) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. .. After the incubation, 1 μl of T4 DNA Polymerase (Thermo Fisher Scientific) was added to the mix, and the mix was further incubated at 25°C for 15 min and at 12°C for 5 min.

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals
    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). .. The USER™ Enzyme mix is fully functional in the PCR buffer and the reaction can be performed directly with the unpurified PCR product reaction.

    Spectrophotometry:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: RNA concentration and purity were measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Concentration Assay:

    Article Title: Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep
    Article Snippet: RNA concentration and purity were measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). .. Then, 3 μl USER Enzyme (NEB, U.S.A.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Marker:

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Correct vectors corresponding to vector pCfB312, bearing an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker (Kl .URA3 ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family, were verified by restriction digest. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

    Variant Assay:

    Article Title: Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
    Article Snippet: The human genes Serpina1 and Serping1 , which encode hα1AT and hC1INH, respectively, either untagged or tagged with S11_M3 or S11_H7 (H7 variant of S11), were subcloned into pcDNA3.1(+)/Zeo (for schematic overview see ; for sequences, see ). .. In brief, PCR amplicons ( ) were made using the proofreading polymerase Pfu X7 and assembled by treating with USER Enzyme (New England Biolabs) and transforming into E. coli One Shot® Mach1™ competent cells (Life Technologies, Thermo Scientific, Rockford, IL).

    Homologous Recombination:

    Article Title: EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
    Article Snippet: Correct vectors corresponding to vector pCfB312, bearing an EasyClone USER cassette and a Kluyveromyces lactis URA3 selection marker (Kl .URA3 ) surrounded by upstream and downstream regions for homologous recombination at LTR from the Ty4 family, were verified by restriction digest. .. After Dpn I treatment, separation and gel-purification, the fragment was circularized on itself in a uracil-excision reaction mediated by the USER enzyme mix (New England Biolabs).

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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