∘ user enzyme  (New England Biolabs)


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    Name:
    USER Enzyme
    Description:
    USER Enzyme 250 units
    Catalog Number:
    M5505L
    Price:
    292
    Size:
    250 units
    Category:
    Other Enzymes
    Score:
    85
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    Name:
    USER Enzyme
    Description:

    Catalog Number:
    M5505S
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs ∘ user enzyme
    USER Enzyme

    https://www.bioz.com/result/∘ user enzyme/product/New England Biolabs
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ∘ user enzyme - by Bioz Stars, 2019-12
    78/100 stars

    Related Products / Commonly Used Together

    pms26 Agilent Technologies

    Images

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1085

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

    Techniques Used: Clone Assay, Sequencing, Construct

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A versatile element for gene addition in bacterial chromosomes
    Article Snippet: Recovery of chromosomal insertions is lower (∼10%) with this element and protocol than the original procedure (50–70%), but faster (4 days from PCR to identified insertion) and more automatable. .. General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions. .. Procedure Day 1: ∘ Step 2 (described above).

    Transformation Assay:

    Article Title: A versatile element for gene addition in bacterial chromosomes
    Article Snippet: Paragraph title: Steps 3–5: USER assembly, transformation and rapid chromosomal insertion ... General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions.

    Construct:

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

    Plasmid Preparation:

    Article Title: A versatile element for gene addition in bacterial chromosomes
    Article Snippet: General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions. .. PCR from template (20 cycles) ∘ Step 3.

    Purification:

    Article Title: A versatile element for gene addition in bacterial chromosomes
    Article Snippet: Recovery of chromosomal insertions is lower (∼10%) with this element and protocol than the original procedure (50–70%), but faster (4 days from PCR to identified insertion) and more automatable. .. General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions. .. Procedure Day 1: ∘ Step 2 (described above).

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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
    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
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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