monarch pcr cleanup kit  (New England Biolabs)


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    Name:
    Monarch PCR and DNA Cleanup Kit
    Description:
    Monarch PCR and DNA Cleanup Kit 250 preps
    Catalog Number:
    t1030l
    Price:
    466
    Size:
    250 preps
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    DNA Cleanup Kits
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    Structured Review

    New England Biolabs monarch pcr cleanup kit
    Monarch PCR and DNA Cleanup Kit
    Monarch PCR and DNA Cleanup Kit 250 preps
    https://www.bioz.com/result/monarch pcr cleanup kit/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    monarch pcr cleanup kit - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

    Amplification:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

    Agarose Gel Electrophoresis:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Ligation:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Isolation:

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

    Purification:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin. .. These reagents recovered 78.1% to 95.7% with 10–50 ng of purified DNA, 81.7% to 96.8% with 5 ng of DNA, and 68.1% to 82.9% with 1 ng of DNA except phenol/chloroform extraction with over 100%.

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments
    Article Snippet: .. Other DNA fragments were obtained by PCR with the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), except for the fragment containing the stem-loop (S-SL), which was obtained with the LA Taq DNA polymerase (Takara Bio Europe) using the GC buffer I. PCR products were purified using either the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim, Germany) or the Monarch™ PCR & DNA Cleanup Kit (NEB). .. Unless otherwise stated, plasmids were obtained from the GeneArt Gene Synthesis Service (Thermo Fisher Scientific).

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Polymerase Chain Reaction:

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy
    Article Snippet: .. The amplification products were purified with Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s recommendations. .. Fragments were sequenced in a capillary electrophoresis instrument AB3730XL (Applied Biosystems).

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin. .. These reagents recovered 78.1% to 95.7% with 10–50 ng of purified DNA, 81.7% to 96.8% with 5 ng of DNA, and 68.1% to 82.9% with 1 ng of DNA except phenol/chloroform extraction with over 100%.

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments
    Article Snippet: .. Other DNA fragments were obtained by PCR with the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), except for the fragment containing the stem-loop (S-SL), which was obtained with the LA Taq DNA polymerase (Takara Bio Europe) using the GC buffer I. PCR products were purified using either the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim, Germany) or the Monarch™ PCR & DNA Cleanup Kit (NEB). .. Unless otherwise stated, plasmids were obtained from the GeneArt Gene Synthesis Service (Thermo Fisher Scientific).

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Gel Extraction:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Chromatin Immunoprecipitation:

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin. .. These reagents recovered 78.1% to 95.7% with 10–50 ng of purified DNA, 81.7% to 96.8% with 5 ng of DNA, and 68.1% to 82.9% with 1 ng of DNA except phenol/chloroform extraction with over 100%.

    Plasmid Preparation:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

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  • 99
    New England Biolabs monarch pcr dna cleanup kit
    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The <t>PCR</t> amplified <t>DNA</t> fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Monarch Pcr Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch pcr dna cleanup kit/product/New England Biolabs
    Average 99 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    monarch pcr dna cleanup kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs monarch dna cleanup kit
    RcGTA gp1 in vitro <t>DNA</t> binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp <t>PCR</t> amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.
    Monarch Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch dna cleanup kit/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    monarch dna cleanup kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Journal: bioRxiv

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    doi: 10.1101/834630

    Figure Lengend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Article Snippet: The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop.

    Techniques: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Sequencing, Polymerase Chain Reaction

    RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.

    Journal: Journal of Virology

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

    doi: 10.1128/JVI.01328-19

    Figure Lengend Snippet: RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.

    Article Snippet: DNA substrates were prepared by PCR amplification with oligonucleotides indicated in and cleaned with a Monarch DNA cleanup kit (NEB).

    Techniques: In Vitro, Binding Assay, Agarose Gel Electrophoresis, Protein Binding, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Amplification, Marker