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vent exo dna polymerase  (New England Biolabs)


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    New England Biolabs vent exo dna polymerase
    Vent Exo Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/vent+exo+polymerase/Vent+(exo/pm41818662-42-5-12
    Average 96 stars, based on 1207 article reviews
    vent exo dna polymerase - by Bioz Stars, 2026-09
    96/100 stars

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    Related Articles

    Polymerase Chain Reaction:

    Article Title: The Impact of Weir Construction in Korea’s Nakdong River on the Population Genetic Variability of the Endangered Fish Species, Rapid Small Gudgeon ( Microphysogobio rapidus )
    Article Snippet: .. PCR was performed in mixtures consisting of 10 μL of DNA, 4 μL of 10 μM SNX primer, 0.3 μL of Vent (-exo) polymerase (New England Biolabs), 4 μL of dNTP mix, 5 μL of 10× Thermopol buffer, and 26.7 μL of sterilized distilled water with denaturation at 96 °C for 5 min followed by 40 cycles of 96 °C for 45 s, 62 °C for 1 min, 72 °C for 2 min, and a final elongation step at 72 °C for 5 min. ..

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: .. PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics).

    Article Title: The Impact of Weir Construction in Korea's Nakdong River on the Population Genetic Variability of the Endangered Fish Species, Rapid Small Gudgeon ( Microphysogobio rapidus ).
    Article Snippet: .. PCR was performed in mixtures consisting of 10 μL of DNA, 4 μL of 10 μM SNX primer, 0.3 μL of Vent (-exo) polymerase (New England Biolabs), 4 μL of dNTP mix, 5 μL of 10× Thermopol buffer, and 26.7 μL of sterilized distilled water with denaturation at 96 ◦C for 5 min followed by 40 cycles of 96 ◦C for 45 s, 62 ◦C for 1 min, 72 ◦C for 2 min, and a final elongation step at 72 ◦C for 5 min. 2.3.3. ..

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics). .. Analysis of Enrichment by DNA Sequencing Using Illumina Sequencing Technology DNA sequencing data was decoded and analyzed as described elsewhere (Petersen et al., “Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library”, Med.

    Purification:

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: .. PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics).

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics). .. Analysis of Enrichment by DNA Sequencing Using Illumina Sequencing Technology DNA sequencing data was decoded and analyzed as described elsewhere (Petersen et al., “Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library”, Med.

    Article Title: Linked-Pair Long-Read Sequencing Strategy for Targeted Resequencing and Enrichment
    Article Snippet: First, 400ng of Cas9nickase -Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1× NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37°C for 15min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37°C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100-300 μM dNTPs, and 1× Thermopol (NEB) at 72°C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. ..

    Article Title: A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.
    Article Snippet: First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 μM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. .. After fragmentation and before library preparation, fragmented and purified DNA samples were assessed by running electrophoresis using a 1% agarose gel slab in 1X TAE buffer at 100 V for 75 min. DNA was stained with 1X SYBRTM Safe stain (Invitrogen) and visualized on UVP GelStudio (Analytik Jena) in epifluorescence mode.

    Introduce:

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: .. PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics).

    Amplification:

    Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression
    Article Snippet: PCR amplification with dideoxynucleotide (ddNTP) termination was conducted using ddNTPs (AAT Bioquest dideoxynucleotides; ddATP, ddTTP, ddGTP, and ddCTP) added individually to separate reactions at a ratio of 1:20 (dNTP:ddNTP). .. Amplification of the sequencing ladders and transcription start site (TSS) mapping via primer extension were performed using Vent (exo-) polymerase following the manufacturer’s protocol (New England Biolabs) with the 5′-Cy5-labeled primers mtnK-rev-Cy5 (fragment 3), Frag2_R 2.0 CY5 (fragment 2), and Frag1_R 2.1 CY5 (fragment 1) ( ). .. Template DNA used for primer extension was prepared from RNA extracted from cells grown as described under Growth Studies and reverse transcribed using Quantabio qScript Ultra SuperMix to create a complementary DNA (cDNA) pool.

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics). .. Analysis of Enrichment by DNA Sequencing Using Illumina Sequencing Technology DNA sequencing data was decoded and analyzed as described elsewhere (Petersen et al., “Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library”, Med.

    Sequencing:

    Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression
    Article Snippet: PCR amplification with dideoxynucleotide (ddNTP) termination was conducted using ddNTPs (AAT Bioquest dideoxynucleotides; ddATP, ddTTP, ddGTP, and ddCTP) added individually to separate reactions at a ratio of 1:20 (dNTP:ddNTP). .. Amplification of the sequencing ladders and transcription start site (TSS) mapping via primer extension were performed using Vent (exo-) polymerase following the manufacturer’s protocol (New England Biolabs) with the 5′-Cy5-labeled primers mtnK-rev-Cy5 (fragment 3), Frag2_R 2.0 CY5 (fragment 2), and Frag1_R 2.1 CY5 (fragment 1) ( ). .. Template DNA used for primer extension was prepared from RNA extracted from cells grown as described under Growth Studies and reverse transcribed using Quantabio qScript Ultra SuperMix to create a complementary DNA (cDNA) pool.

    Article Title: A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment
    Article Snippet: First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 μM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. .. After fragmentation and before library preparation, fragmented and purified DNA samples were assessed by running electrophoresis using a 1% agarose gel slab in 1X TAE buffer at 100 V for 75 min. DNA was stained with 1X SYBRTM Safe stain (Invitrogen) and visualized on UVP GelStudio (Analytik Jena) in epifluorescence mode.

    Article Title: Linked-Pair Long-Read Sequencing Strategy for Targeted Resequencing and Enrichment
    Article Snippet: First, 400ng of Cas9nickase -Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1× NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37°C for 15min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37°C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100-300 μM dNTPs, and 1× Thermopol (NEB) at 72°C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. ..

    Article Title: A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.
    Article Snippet: First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 μM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. .. After fragmentation and before library preparation, fragmented and purified DNA samples were assessed by running electrophoresis using a 1% agarose gel slab in 1X TAE buffer at 100 V for 75 min. DNA was stained with 1X SYBRTM Safe stain (Invitrogen) and visualized on UVP GelStudio (Analytik Jena) in epifluorescence mode.

    DNA Sequencing:

    Article Title: Method for screening of an in vitro display library within a cell
    Article Snippet: PCR Mixture (Final Concentrations): 25 μl purified ligated DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.4 mM CleanAmp dNTP (tebu-bio) 6 mM MgCl2 (Sigma-Aldrich) 3% DMSO (Sigma-Aldrich) 0.25 μM forward PCR primer 0.25 μM backward PCR primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 95° C. 10 min, 27 cycles of (95° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified on NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel), and subjected to a second round of PCR to introduce priming sites for Illumina sequencing. .. PCR mixture: 5 μl purified amplified DNA (PCR template) 1× Vent (-exo) PCR buffer (New England Biolabs) 0.2 mM dNTP (Thermo Fisher Scientific) 6 mM MgCl2 (Sigma-Aldrich) 0.5M betaine (Sigma-Aldrich) 0.25 μM forward Illumina primer 0.25 μM backward Illumina primer 1 U Vent (-exo) polymerase (New England Biolabs) Water to a total of 100 μl The mixture was subjected to thermal cycling by applying the following program in a PCR machine: 92° C. 2 min, 10 cycles of (92° C. 30 sec, 62° C. 30 sec, 72° C. 2 min30 sec), 72° C. 2 min The resulting library of DNA fragments was purified by Agencourt AMPure XP (Beckman Coulter Life Sciences) and preparative 10% TBE-PAGE (Kem-En-Tec) and submitted for Illumina DNA sequencing (Fulgent Genetics). .. Analysis of Enrichment by DNA Sequencing Using Illumina Sequencing Technology DNA sequencing data was decoded and analyzed as described elsewhere (Petersen et al., “Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library”, Med.

    Agarose Gel Electrophoresis:

    Article Title: A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment
    Article Snippet: First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 μM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. .. After fragmentation and before library preparation, fragmented and purified DNA samples were assessed by running electrophoresis using a 1% agarose gel slab in 1X TAE buffer at 100 V for 75 min. DNA was stained with 1X SYBRTM Safe stain (Invitrogen) and visualized on UVP GelStudio (Analytik Jena) in epifluorescence mode.

    Article Title: Linked-Pair Long-Read Sequencing Strategy for Targeted Resequencing and Enrichment
    Article Snippet: First, 400ng of Cas9nickase -Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1× NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37°C for 15min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37°C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100-300 μM dNTPs, and 1× Thermopol (NEB) at 72°C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. ..

    Article Title: A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.
    Article Snippet: First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. .. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 μM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation. .. After fragmentation and before library preparation, fragmented and purified DNA samples were assessed by running electrophoresis using a 1% agarose gel slab in 1X TAE buffer at 100 V for 75 min. DNA was stained with 1X SYBRTM Safe stain (Invitrogen) and visualized on UVP GelStudio (Analytik Jena) in epifluorescence mode.



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