e coli dna ligase  (New England Biolabs)


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    Name:
    E coli DNA Ligase
    Description:
    E coli DNA Ligase 1 000 units
    Catalog Number:
    m0205l
    Price:
    248
    Size:
    1 000 units
    Category:
    DNA Ligases
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    New England Biolabs e coli dna ligase
    E coli DNA Ligase
    E coli DNA Ligase 1 000 units
    https://www.bioz.com/result/e coli dna ligase/product/New England Biolabs
    Average 95 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    e coli dna ligase - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

    The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.
    Figure Legend Snippet: The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

    Techniques Used: Plasmid Preparation, Incubation, Activity Assay

    ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.
    Figure Legend Snippet: ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.

    Techniques Used: Activity Assay, Incubation, Agarose Gel Electrophoresis, Labeling

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
    Figure Legend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Techniques Used: Incubation, Plasmid Preparation, Labeling

    2) Product Images from "Deoxyinosine repair in nuclear extracts of human cells"

    Article Title: Deoxyinosine repair in nuclear extracts of human cells

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-015-0044-8

    Map of M13mp18 and f1PM based heteroduplex substrates. a The map of bacteriophage M13mp18 replicative form (RF) DNA shows restriction enzyme sites relevant to this study with derivatives M13LR1 and M13LR3 containing 22-bp insertions at the unique HindIII restriction site, and phage M13WX1 and M13X22 containing 26-bp and 22-bp insertions at Xba I site respectively. b The map of bacteriophage f1PM RF DNA with its derivative f1PMA with a 27-bp insertion at Xba I. ‘V’, phage viral strand. ‘C’, phage complementary strand. Underlines beneath each viral strand are the original insertion sequences. The C-strand from parental phage RF DNA was paired with viral strand of its insertion derivative to produce gapped duplex DNA, and the gap was sealed with dI or deoxyuridine containing synthetic oligodeoxyribonucleotide. A-I, C-I, G-I, T-I, and G-U are the resulting substrates and DNA sequence shown on each C-strand of the the synthetic linker used. In the presence of dI, the substrates were refractory to the restriction endonuclease scoring. After the repair, DNA products become sensitive to restriction endonuclease cleavage. The recognition sequence of corresponding restriction endonuclease markers for repair products are shown in bold on V-strands
    Figure Legend Snippet: Map of M13mp18 and f1PM based heteroduplex substrates. a The map of bacteriophage M13mp18 replicative form (RF) DNA shows restriction enzyme sites relevant to this study with derivatives M13LR1 and M13LR3 containing 22-bp insertions at the unique HindIII restriction site, and phage M13WX1 and M13X22 containing 26-bp and 22-bp insertions at Xba I site respectively. b The map of bacteriophage f1PM RF DNA with its derivative f1PMA with a 27-bp insertion at Xba I. ‘V’, phage viral strand. ‘C’, phage complementary strand. Underlines beneath each viral strand are the original insertion sequences. The C-strand from parental phage RF DNA was paired with viral strand of its insertion derivative to produce gapped duplex DNA, and the gap was sealed with dI or deoxyuridine containing synthetic oligodeoxyribonucleotide. A-I, C-I, G-I, T-I, and G-U are the resulting substrates and DNA sequence shown on each C-strand of the the synthetic linker used. In the presence of dI, the substrates were refractory to the restriction endonuclease scoring. After the repair, DNA products become sensitive to restriction endonuclease cleavage. The recognition sequence of corresponding restriction endonuclease markers for repair products are shown in bold on V-strands

    Techniques Used: Sequencing

    3) Product Images from "Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A"

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.7.2037-2046.2002

    Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.
    Figure Legend Snippet: Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.

    Techniques Used: In Vitro, Fractionation, Incubation, Agarose Gel Electrophoresis, Staining

    4) Product Images from "Independent and Stochastic Action of DNA Polymerases in the Replisome"

    Article Title: Independent and Stochastic Action of DNA Polymerases in the Replisome

    Journal: Cell

    doi: 10.1016/j.cell.2017.05.041

    Individual replication fork progression is independent of primase A Micrographs showing replication products at 10 min where: i , all components present; or a component omitted: ii, DnaB and DnaC810; iii, Pol III*; iv, β; v, SSB; vi, primase. Composite, false-colored fields show anchor points for molecules that contain ssDNA, except i or v , where only long products were seen. In vi , surfaces were sparsely populated with DNA to avoid any ambiguity in molecule identification. Cyan, fields with flow off; magenta, same field with flow on showing fully-extended molecules. Molecules are bracketed for clarity. Scale bar: 10 μm, equal to 33.9 kb dsDNA or 80.3 knt SSB-bound ssDNA at 4,000 μl/h, without Mg 2+ ). B. Cartoon showing leading strand only product in a reaction lacking primase. C. Composite, false-colored image showing leading strand only replication without primase. Three replicating molecules ( 1, 2, 3 ). Molecules a, b and c are referred to later. D. Time-lapse, at 50-second intervals, of Molecules 1, 2 and 3 identified in C , colored by time-point as per C . E. Kymographs of molecules, numbered per C and D , showing fork progression without primase. Dashed grey line: position of anchor. Linear fits are from initiation to termination, yielding average fork rates. Pauses are included in the average here. F. Histograms of fork progression rates in the presence (grey) and absence of primase (light blue). Histograms fit to single Gaussians ( R 2 : with primase, 0.80; without primase, 0.94); no outliers were rejected. n , molecules. G. Processivities of single replisomes from live imaging experiments. Whisker plots of molecule lengths, with (320 nM) or without primase and/or β in flow. Data from 2 (primase, no β) or 3 (others) experiments. Horizontal bars, median; vertical bars, interquartile range. ***, significantly different pairs of populations (Kruskal-Wallis; P
    Figure Legend Snippet: Individual replication fork progression is independent of primase A Micrographs showing replication products at 10 min where: i , all components present; or a component omitted: ii, DnaB and DnaC810; iii, Pol III*; iv, β; v, SSB; vi, primase. Composite, false-colored fields show anchor points for molecules that contain ssDNA, except i or v , where only long products were seen. In vi , surfaces were sparsely populated with DNA to avoid any ambiguity in molecule identification. Cyan, fields with flow off; magenta, same field with flow on showing fully-extended molecules. Molecules are bracketed for clarity. Scale bar: 10 μm, equal to 33.9 kb dsDNA or 80.3 knt SSB-bound ssDNA at 4,000 μl/h, without Mg 2+ ). B. Cartoon showing leading strand only product in a reaction lacking primase. C. Composite, false-colored image showing leading strand only replication without primase. Three replicating molecules ( 1, 2, 3 ). Molecules a, b and c are referred to later. D. Time-lapse, at 50-second intervals, of Molecules 1, 2 and 3 identified in C , colored by time-point as per C . E. Kymographs of molecules, numbered per C and D , showing fork progression without primase. Dashed grey line: position of anchor. Linear fits are from initiation to termination, yielding average fork rates. Pauses are included in the average here. F. Histograms of fork progression rates in the presence (grey) and absence of primase (light blue). Histograms fit to single Gaussians ( R 2 : with primase, 0.80; without primase, 0.94); no outliers were rejected. n , molecules. G. Processivities of single replisomes from live imaging experiments. Whisker plots of molecule lengths, with (320 nM) or without primase and/or β in flow. Data from 2 (primase, no β) or 3 (others) experiments. Horizontal bars, median; vertical bars, interquartile range. ***, significantly different pairs of populations (Kruskal-Wallis; P

    Techniques Used: Flow Cytometry, Imaging, Whisker Assay

    Related Articles

    Clone Assay:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Paragraph title: cDNA cloning of recombinant His-, GFP-, or RFP-tagged proteins ... Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA.

    Amplification:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. The coding sequences of various genes (see ) were amplified by PCR from liver cDNA using Advantage® cDNA Polymerase Mix (BD Biosciences Clontech, Palo Alto, CA).

    Article Title: PI3K signaling and miRNA expression during the response of quiescent human fibroblasts to distinct proliferative stimuli
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    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Twenty different indexes were added to individual libraries using Phusion DNA polymerase (New England Biolabs) during 16 cycles of amplification.

    DNA Synthesis:

    Article Title: Emerging recombinant noroviruses identified by clinical and waste water screening
    Article Snippet: .. First-strand cDNA was prepared using Protoscript II kit (New England Biolabs, Massachusetts, USA) followed by second-strand DNA synthesis using a cocktail of Escherichia coli DNA ligase, DNA polymerase I and RNase H (NEB). .. Nextera XT library preparation for NGS Illumina libraries were prepared using the Nextera XT DNA sample preparation kit and quantified using the Quant-iT PicoGreen dsDNA assay kit.

    DNA Ligation:

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Finally, libraries were prepared using End-It DNA End Repair Kit (Epicentre, Madison, WI, USA) and Klenow Fragment (3′→5′ exo-) (New England Biolabs) for the ligation of Illumina sequencing adapters (Illumina-Index-AdapterA and 5’-phosphorylated Illumina-Index-AdapterB) using the LigaFast™ Rapid DNA Ligation System (Promega, Madison, WI, USA).

    Synthesized:

    Article Title: Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response
    Article Snippet: First-strand cDNA was synthesized using the SuperScript™ First-strand Synthesis System for reverse transcription-PCR (RT-PCR; Invitrogen, Carlsbad, CA, USA) and oligo(dT)12–18 (Promega, Madison, WI, USA). .. Second-strand synthesis was performed in second-strand synthesis buffer (20 mM TRIS-HCl, pH 7.5, 100 mM KCl, 10 mM NH4 SO4 ) by adding 5 mM MgCl2 , 0.15 mM β-NAD+ , 0.5 mg ml−1 bovine serum albumin (BSA), 0.2 mM dNTPs, 10 μl of template single-stranded cDNA, 6 U of DNA polymerase I, 0.25 U of RNase-H, and 1.2 U of Escherichia coli DNA ligase (New England BioLabs, Ipswich, MA, USA).

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: .. PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were biotin labeled using the Bioprime system (Invitrogen) and concentrated by ethanol precipitation.

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
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    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
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    Article Title: Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations
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    Electrophoresis:

    Article Title: The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops
    Article Snippet: To assay the introduction of negative supercoils, 26 μ m NAD and E. coli DNA ligase (New England Biolabs, 0.4 units) were included in the DNA condensation reaction mixtures that were incubated for 30 min at 37 °C. .. DNA products were resolved by electrophoresis through 0.8% agarose gels at 30 V for 15 h at room temperature using 50 m m Tris-HCl (pH 7.8 at 23 °C), 40 m m NaOAc, 1 m m EDTA, and 10 μg/ml chloroquine di-phosphate as the running buffer.

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    Microarray:

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    Incubation:

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    Article Snippet: .. To assay the introduction of negative supercoils, 26 μ m NAD and E. coli DNA ligase (New England Biolabs, 0.4 units) were included in the DNA condensation reaction mixtures that were incubated for 30 min at 37 °C. .. Reactions were stopped by the addition of EDTA and KCl to final concentrations of 50 and 300 m m , respectively, followed by incubation for 5 min at room temperature.

    Article Title: Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response
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    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: .. PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were biotin labeled using the Bioprime system (Invitrogen) and concentrated by ethanol precipitation.

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: .. Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. The coding sequences of various genes (see ) were amplified by PCR from liver cDNA using Advantage® cDNA Polymerase Mix (BD Biosciences Clontech, Palo Alto, CA).

    Article Title: PI3K signaling and miRNA expression during the response of quiescent human fibroblasts to distinct proliferative stimuli
    Article Snippet: .. After 2 hours incubation, 2nd strand cDNA synthesis was carried out by adding 2.5 μl Escherichia coli DNA polymerase I (10 U/μl; NEB, Beverly, MA, USA), 1.5 μl E. coli DNA ligase (10 U/μl; NEB), 1 μl RNase H (10 U/μl; Ambion, Austin, TX, USA), 3 μl of 10 mM dNTP mix, 10 μl of 10x 2nd strand synthesis buffer, and 62 μl DEPC-treated water. ..

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: The first strand reaction was incubated at 25 °C for 10 min, and 50 °C for 90 min, followed by deactivation at 70 °C for 15 min, after which the MinElute Reaction Cleanup Kit (Qiagen) was used according to the manufacturer's protocol. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Article Title: Integrated transcriptome analysis of the cellular mechanisms associated with Ha-ras-dependent malignant transformation of the human breast epithelial MCF7 cell line
    Article Snippet: .. The first strand samples were then added to 170 µl aliquots of diluted second strand synthesis mix [10× Escherichia coli DNA ligase buffer (New England Biolabs), 900 mM KCl (Sigma), 20 mg/ml glycogen (Boehringer), 10 mM dNTP each, 10 U/µl E.coli DNA ligase (NEB), 2 U/µl Rnase H and 10 U/µl E.coli DNA polymerase I, all purchased from Promega] and incubated for 2 h at 16°C in a Perkin Elmer 9700 thermal cycler. .. The cDNA populations, still bound to Dynabeads, were then digested with Sau3A (4 U/µl New England Biolabs) for 2 h at 37°C, generating fragments of about 256 bp in statistical length, then the enzyme was inactivated for 20 min at 65°C.

    Activity Assay:

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
    Article Snippet: DNA ligase activity in FII was determined by its ability to convert a nicked circular substrate into supercoiled DNA. .. As a positive control, the nicked DNA substrate was also incubated with E. coli DNA ligase (New England Biolabs) at a final concentration of 4 U/μg of DNA.

    Expressing:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. Respective primers were designed to create 5′ and 3′ restriction endonuclease cleavage sites for in-frame ligation into expression vector pcDNA4/HisMax (Invitrogen™).

    Modification:

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: Sequencing libraries for whole transcriptome analysis were generated using a modified Illumina TruSeq sample preparation protocol. .. After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs).

    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
    Article Snippet: First strand cDNA was synthesized using ProtoScript M-MuLV FS-cDNA Synthesis Kit (New England Biolabs, MA) according to the manufacturer’s protocol and modified oligonucleotides in Supporting Information , Table S1 . .. Second strand synthesis was performed by incubating first-strand cDNA with 1× NEBNext Second Strand Synthesis Buffer (New England Biolabs, MA), 0.2 mM dNTPs, 15 units of Escherichia coli DNA ligase (New England Biolabs, MA), 75 units of E. coli DNA polymerase I (New England Biolabs, MA), and 3 units of RNase H (New England Biolabs, MA) for 2 hr at 16°. cDNA was purified using the GeneJet PCR Purification Kit (Fermentas, MA) and then fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, MA) according to the manufacturer’s protocol, with the addition of 5 mM MgCl2 and 1 mg ml−1 BSA (New England Biolabs, MA).

    Positive Control:

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
    Article Snippet: .. As a positive control, the nicked DNA substrate was also incubated with E. coli DNA ligase (New England Biolabs) at a final concentration of 4 U/μg of DNA. .. DNA samples were recovered, fractionated in a 1% agarose gel, and visualized under UV illumination.

    Ligation:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. Respective primers were designed to create 5′ and 3′ restriction endonuclease cleavage sites for in-frame ligation into expression vector pcDNA4/HisMax (Invitrogen™).

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Finally, libraries were prepared using End-It DNA End Repair Kit (Epicentre, Madison, WI, USA) and Klenow Fragment (3′→5′ exo-) (New England Biolabs) for the ligation of Illumina sequencing adapters (Illumina-Index-AdapterA and 5’-phosphorylated Illumina-Index-AdapterB) using the LigaFast™ Rapid DNA Ligation System (Promega, Madison, WI, USA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response
    Article Snippet: First-strand cDNA was synthesized using the SuperScript™ First-strand Synthesis System for reverse transcription-PCR (RT-PCR; Invitrogen, Carlsbad, CA, USA) and oligo(dT)12–18 (Promega, Madison, WI, USA). .. Second-strand synthesis was performed in second-strand synthesis buffer (20 mM TRIS-HCl, pH 7.5, 100 mM KCl, 10 mM NH4 SO4 ) by adding 5 mM MgCl2 , 0.15 mM β-NAD+ , 0.5 mg ml−1 bovine serum albumin (BSA), 0.2 mM dNTPs, 10 μl of template single-stranded cDNA, 6 U of DNA polymerase I, 0.25 U of RNase-H, and 1.2 U of Escherichia coli DNA ligase (New England BioLabs, Ipswich, MA, USA).

    Article Title: Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations
    Article Snippet: RNA for tiling arrays, RT-PCR, and RNA sequencing was prepared at each generation. .. Second-strand cDNA for tiling array was synthesized by using Second Strand Buffer (Invitrogen), E. coli DNA ligase (NEB), E. coli DNA polymerase I (Promega), and T4 DNA polymerase (NEB).

    Generated:

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: .. After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs). .. The double-stranded cDNAs were sheared to ∼150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis.

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: .. First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Finally, libraries were prepared using End-It DNA End Repair Kit (Epicentre, Madison, WI, USA) and Klenow Fragment (3′→5′ exo-) (New England Biolabs) for the ligation of Illumina sequencing adapters (Illumina-Index-AdapterA and 5’-phosphorylated Illumina-Index-AdapterB) using the LigaFast™ Rapid DNA Ligation System (Promega, Madison, WI, USA).

    other:

    Article Title: RecN Is a Cohesin-like Protein That Stimulates Intermolecular DNA Interactions in Vitro *
    Article Snippet: All of the restriction endonucleases and E. coli DNA ligase were purchased from New England Biolabs.

    cDNA-AFLP Assay:

    Article Title: Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response
    Article Snippet: Paragraph title: Preparation of poly(A)+ RNA and cDNA synthesis for cDNA-AFLP experiments ... Second-strand synthesis was performed in second-strand synthesis buffer (20 mM TRIS-HCl, pH 7.5, 100 mM KCl, 10 mM NH4 SO4 ) by adding 5 mM MgCl2 , 0.15 mM β-NAD+ , 0.5 mg ml−1 bovine serum albumin (BSA), 0.2 mM dNTPs, 10 μl of template single-stranded cDNA, 6 U of DNA polymerase I, 0.25 U of RNase-H, and 1.2 U of Escherichia coli DNA ligase (New England BioLabs, Ipswich, MA, USA).

    Sequencing:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP. .. Libraries were enriched in a 10-cycle PCR with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) and pooled in equimolar ratios for multiplex sequencing.

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: Sequencing libraries for whole transcriptome analysis were generated using a modified Illumina TruSeq sample preparation protocol. .. After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs).

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Finally, libraries were prepared using End-It DNA End Repair Kit (Epicentre, Madison, WI, USA) and Klenow Fragment (3′→5′ exo-) (New England Biolabs) for the ligation of Illumina sequencing adapters (Illumina-Index-AdapterA and 5’-phosphorylated Illumina-Index-AdapterB) using the LigaFast™ Rapid DNA Ligation System (Promega, Madison, WI, USA).

    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
    Article Snippet: Paragraph title: Preparation of sequencing libraries ... Second strand synthesis was performed by incubating first-strand cDNA with 1× NEBNext Second Strand Synthesis Buffer (New England Biolabs, MA), 0.2 mM dNTPs, 15 units of Escherichia coli DNA ligase (New England Biolabs, MA), 75 units of E. coli DNA polymerase I (New England Biolabs, MA), and 3 units of RNase H (New England Biolabs, MA) for 2 hr at 16°. cDNA was purified using the GeneJet PCR Purification Kit (Fermentas, MA) and then fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, MA) according to the manufacturer’s protocol, with the addition of 5 mM MgCl2 and 1 mg ml−1 BSA (New England Biolabs, MA).

    Article Title: Emerging recombinant noroviruses identified by clinical and waste water screening
    Article Snippet: Paragraph title: Full-length norovirus genome sequencing preparation ... First-strand cDNA was prepared using Protoscript II kit (New England Biolabs, Massachusetts, USA) followed by second-strand DNA synthesis using a cocktail of Escherichia coli DNA ligase, DNA polymerase I and RNase H (NEB).

    Sonication:

    Article Title: Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations
    Article Snippet: Second-strand cDNA for tiling array was synthesized by using Second Strand Buffer (Invitrogen), E. coli DNA ligase (NEB), E. coli DNA polymerase I (Promega), and T4 DNA polymerase (NEB). .. Synthesized cDNA were fragmented by sonication for tiling arrays.

    Recombinant:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Paragraph title: cDNA cloning of recombinant His-, GFP-, or RFP-tagged proteins ... Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA.

    DNA Gel Electrophoresis:

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs). .. The double-stranded cDNAs were sheared to ∼150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis.

    Nucleic Acid Electrophoresis:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP. .. Illumina adapters with four base barcodes were ligated to cDNA and fragments ranging from 150-250 bp were selected using gel electrophoresis.

    Multiplex Assay:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP. .. Libraries were enriched in a 10-cycle PCR with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) and pooled in equimolar ratios for multiplex sequencing.

    RNA Sequencing Assay:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: Paragraph title: RNA-Sequencing ... First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP.

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: Paragraph title: RNA sequencing and analysis ... After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs).

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: Paragraph title: RNA Sequencing analyses ... First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies).

    Article Title: Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations
    Article Snippet: RNA for tiling arrays, RT-PCR, and RNA sequencing was prepared at each generation. .. Second-strand cDNA for tiling array was synthesized by using Second Strand Buffer (Invitrogen), E. coli DNA ligase (NEB), E. coli DNA polymerase I (Promega), and T4 DNA polymerase (NEB).

    Isolation:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Poly A+ RNA was isolated from liver total RNA using the Oligotex® mRNA Mini Kit from Qiagen GmbH (Hilden, Germany). .. Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA.

    Article Title: PI3K signaling and miRNA expression during the response of quiescent human fibroblasts to distinct proliferative stimuli
    Article Snippet: Paragraph title: RNA isolation and amplification ... After 2 hours incubation, 2nd strand cDNA synthesis was carried out by adding 2.5 μl Escherichia coli DNA polymerase I (10 U/μl; NEB, Beverly, MA, USA), 1.5 μl E. coli DNA ligase (10 U/μl; NEB), 1 μl RNase H (10 U/μl; Ambion, Austin, TX, USA), 3 μl of 10 mM dNTP mix, 10 μl of 10x 2nd strand synthesis buffer, and 62 μl DEPC-treated water.

    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
    Article Snippet: Preparation of sequencing libraries Polyadenylated RNA was purified from 10 µg of total RNA using the Magnetic mRNA Isolation Kit (New England Biolabs, MA). .. Second strand synthesis was performed by incubating first-strand cDNA with 1× NEBNext Second Strand Synthesis Buffer (New England Biolabs, MA), 0.2 mM dNTPs, 15 units of Escherichia coli DNA ligase (New England Biolabs, MA), 75 units of E. coli DNA polymerase I (New England Biolabs, MA), and 3 units of RNase H (New England Biolabs, MA) for 2 hr at 16°. cDNA was purified using the GeneJet PCR Purification Kit (Fermentas, MA) and then fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, MA) according to the manufacturer’s protocol, with the addition of 5 mM MgCl2 and 1 mg ml−1 BSA (New England Biolabs, MA).

    RNA Extraction:

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: Paragraph title: RNA extraction ... The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Article Title: Emerging recombinant noroviruses identified by clinical and waste water screening
    Article Snippet: RNA was extracted from a 10% (v/v) faecal suspension using QIAamp viral RNA extraction kit (Qiagen). .. First-strand cDNA was prepared using Protoscript II kit (New England Biolabs, Massachusetts, USA) followed by second-strand DNA synthesis using a cocktail of Escherichia coli DNA ligase, DNA polymerase I and RNase H (NEB).

    Labeling:

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were biotin labeled using the Bioprime system (Invitrogen) and concentrated by ethanol precipitation.

    Purification:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: The reaction was quenched by the addition of 2 μl volumes of 200 mM EDTA and purified with an Illustra Microspin G25 column (GE Healthcare). .. First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP.

    Article Title: Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response
    Article Snippet: Poly(A)+ RNA was prepared using a Dynabeads® mRNA Purification Kit (Dynal, Oslo, Norway) according to the manufacturer's instructions. .. Second-strand synthesis was performed in second-strand synthesis buffer (20 mM TRIS-HCl, pH 7.5, 100 mM KCl, 10 mM NH4 SO4 ) by adding 5 mM MgCl2 , 0.15 mM β-NAD+ , 0.5 mg ml−1 bovine serum albumin (BSA), 0.2 mM dNTPs, 10 μl of template single-stranded cDNA, 6 U of DNA polymerase I, 0.25 U of RNase-H, and 1.2 U of Escherichia coli DNA ligase (New England BioLabs, Ipswich, MA, USA).

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: .. PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were biotin labeled using the Bioprime system (Invitrogen) and concentrated by ethanol precipitation.

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB). .. After 2 h at 16 °C, 1 μl of T4 DNA polymerase (10 U/μl Promega) was added and the reaction was incubated at 16 °C for 10 min.

    Article Title: Integrated transcriptome analysis of the cellular mechanisms associated with Ha-ras-dependent malignant transformation of the human breast epithelial MCF7 cell line
    Article Snippet: The first strand samples were then added to 170 µl aliquots of diluted second strand synthesis mix [10× Escherichia coli DNA ligase buffer (New England Biolabs), 900 mM KCl (Sigma), 20 mg/ml glycogen (Boehringer), 10 mM dNTP each, 10 U/µl E.coli DNA ligase (NEB), 2 U/µl Rnase H and 10 U/µl E.coli DNA polymerase I, all purchased from Promega] and incubated for 2 h at 16°C in a Perkin Elmer 9700 thermal cycler. .. The PCR products were purified using a Qiaquick PCR purification kit (Qiagen) and utilized for VGID™ or micro-array experiments (see below).

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: Briefly, Poly A RNA enrichment was performed using Dynabeads mRNA Purification Kit (Life Technologies, Carlsbad, CA, USA) and mRNA fragmentation using a fragmentation buffer (40 mM Tris acetate, 100 mM K acetate, 30 mM Mg acetate, pH 8.2; 2 min 30 s at 95 C). .. First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies).

    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
    Article Snippet: .. Second strand synthesis was performed by incubating first-strand cDNA with 1× NEBNext Second Strand Synthesis Buffer (New England Biolabs, MA), 0.2 mM dNTPs, 15 units of Escherichia coli DNA ligase (New England Biolabs, MA), 75 units of E. coli DNA polymerase I (New England Biolabs, MA), and 3 units of RNase H (New England Biolabs, MA) for 2 hr at 16°. cDNA was purified using the GeneJet PCR Purification Kit (Fermentas, MA) and then fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, MA) according to the manufacturer’s protocol, with the addition of 5 mM MgCl2 and 1 mg ml−1 BSA (New England Biolabs, MA). .. Fragmented cDNA was purified and the ends were repaired using NEB Quick Blunting Kit (New England Biolabs, MA) according to manufacturer’s protocol.

    Article Title: Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations
    Article Snippet: Mixed staged animals were collected with M9 buffer, and total RNA was purified by a guanidinium thiocyanate-phenol-chloroform (TRIzol) extraction method ( ). .. Second-strand cDNA for tiling array was synthesized by using Second Strand Buffer (Invitrogen), E. coli DNA ligase (NEB), E. coli DNA polymerase I (Promega), and T4 DNA polymerase (NEB).

    Polymerase Chain Reaction:

    Article Title: Reduced DICER1 elicits an interferon response in endometrial cancer cells
    Article Snippet: First strand cDNA was made using hexameric random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), and the product was treated with E. coli DNA ligase, DNA polymerase I, and RNase H to prepare double stranded cDNA using standard methods. cDNA libraries were end-repaired with a Quick Blunting kit (New England BioLabs, Ipswich, MA) and A-tailed using Klenow exo- and dATP. .. Libraries were enriched in a 10-cycle PCR with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) and pooled in equimolar ratios for multiplex sequencing.

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. The coding sequences of various genes (see ) were amplified by PCR from liver cDNA using Advantage® cDNA Polymerase Mix (BD Biosciences Clontech, Palo Alto, CA).

    Article Title: Integrated transcriptome analysis of the cellular mechanisms associated with Ha-ras-dependent malignant transformation of the human breast epithelial MCF7 cell line
    Article Snippet: The first strand samples were then added to 170 µl aliquots of diluted second strand synthesis mix [10× Escherichia coli DNA ligase buffer (New England Biolabs), 900 mM KCl (Sigma), 20 mg/ml glycogen (Boehringer), 10 mM dNTP each, 10 U/µl E.coli DNA ligase (NEB), 2 U/µl Rnase H and 10 U/µl E.coli DNA polymerase I, all purchased from Promega] and incubated for 2 h at 16°C in a Perkin Elmer 9700 thermal cycler. .. The PCR products were purified using a Qiaquick PCR purification kit (Qiagen) and utilized for VGID™ or micro-array experiments (see below).

    Article Title: De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes
    Article Snippet: .. Second strand synthesis was performed by incubating first-strand cDNA with 1× NEBNext Second Strand Synthesis Buffer (New England Biolabs, MA), 0.2 mM dNTPs, 15 units of Escherichia coli DNA ligase (New England Biolabs, MA), 75 units of E. coli DNA polymerase I (New England Biolabs, MA), and 3 units of RNase H (New England Biolabs, MA) for 2 hr at 16°. cDNA was purified using the GeneJet PCR Purification Kit (Fermentas, MA) and then fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, MA) according to the manufacturer’s protocol, with the addition of 5 mM MgCl2 and 1 mg ml−1 BSA (New England Biolabs, MA). .. Fragmented cDNA was purified and the ends were repaired using NEB Quick Blunting Kit (New England Biolabs, MA) according to manufacturer’s protocol.

    Agarose Gel Electrophoresis:

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
    Article Snippet: As a positive control, the nicked DNA substrate was also incubated with E. coli DNA ligase (New England Biolabs) at a final concentration of 4 U/μg of DNA. .. DNA samples were recovered, fractionated in a 1% agarose gel, and visualized under UV illumination.

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Fragments between 150-300 bp were recovered after 2% agarose gel electrophoresis in order to remove adapter dimers by size selection (QIAquick Gel Extraction Kit, Qiagen).

    Chromatin Immunoprecipitation:

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: The quantity and quality of RNA samples was determined using BioAnalyzer and RNA Nano Lab Chip Kit (Agilent Technologies, Santa Clara, CA, USA). .. First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies).

    Plasmid Preparation:

    Article Title: Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver
    Article Snippet: Second-strand cDNA was obtained from first-strand cDNA by addition of Escherichia coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs, Inc., Beverly, MA), and deoxyribonucleotide triphosphates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16°C for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs) was added and further incubated for 20 min to give blunt end cDNA. .. Respective primers were designed to create 5′ and 3′ restriction endonuclease cleavage sites for in-frame ligation into expression vector pcDNA4/HisMax (Invitrogen™).

    Functional Assay:

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were hybridized to Genechip Arabidopsis Tiling 1.0F arrays (Affymetrix) and probe intensities were scanned at the University of Chicago Functional Genomics Center.

    Selection:

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Fragments between 150-300 bp were recovered after 2% agarose gel electrophoresis in order to remove adapter dimers by size selection (QIAquick Gel Extraction Kit, Qiagen).

    Sample Prep:

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: Sequencing libraries for whole transcriptome analysis were generated using a modified Illumina TruSeq sample preparation protocol. .. After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs).

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: Paragraph title: Whole-genome tiling array sample preparation and signal normalization. ... PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen).

    Column Chromatography:

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
    Article Snippet: As a positive control, the nicked DNA substrate was also incubated with E. coli DNA ligase (New England Biolabs) at a final concentration of 4 U/μg of DNA. .. Reactions were terminated by addition of EDTA to a final concentration of 20 mM, and unincorporated nucleotides were removed by Sephadex G-50 spin column chromatography.

    Ethanol Precipitation:

    Article Title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
    Article Snippet: PolyA RNA was purified with Oligotex beads (Qiagen), and random hexamer-primed first-strand cDNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 42 °C for 1 h. Second-strand cDNA was synthesized in second-strand reaction buffer (Invitrogen) with 40 U of E. coli DNA polymerase I (New England Biolabs), 10 U of E. coli DNA ligase (New England Biolabs), and 2 U of E. coli RNase H (Epicentre) at 16 °C for 2 h. cDNA samples were incubated with 10 U of RNase H, 0.5 U of RNaseA, and 20 U of RNaseT1 at 37 °C for 20 min and then purified on Qiaquick spin columns (Qiagen). .. Samples were biotin labeled using the Bioprime system (Invitrogen) and concentrated by ethanol precipitation.

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: Each 50 μl fragmentation reaction was incubated at 95 °C for 1.5 min on a thermal cycler, and placed on ice for 10 min. Ethanol precipitation was used to purify the reactions. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Random Hexamer Labeling:

    Article Title: ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
    Article Snippet: A Superscript III Reverse Transcriptase First-Strand Synthesis (Invitrogen) system was used to generate cDNA from the polyA selected RNA with random hexamer primers (Invitrogen). .. After first strand synthesis a second strand was generated using Escherichia coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs).

    Concentration Assay:

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
    Article Snippet: .. As a positive control, the nicked DNA substrate was also incubated with E. coli DNA ligase (New England Biolabs) at a final concentration of 4 U/μg of DNA. .. DNA samples were recovered, fractionated in a 1% agarose gel, and visualized under UV illumination.

    Article Title: Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Article Snippet: The RNA fragmentation reactions were performed using fragmentation buffer (5 ×; 200 mM Tris-Ac, 500 mM Potassium-Ac, 150 mM Magnesium-Ac) in a final concentration of 1 × per reaction. .. The second strand was synthesized by adding to the purified sample (100 μl total volume): 20 μl 5 × Second strand buffer (Invitrogen), 4 μl 5 × First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8 U/ml Ambion), 1 μl E scherichia coli DNA polymerase I (10 U/μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB).

    Gel Extraction:

    Article Title: Ovarian transcriptome associated with reproductive senescence in the long-living Ames dwarf mice
    Article Snippet: First strand cDNA was generated using Superscript III 1st Strand Synthesis System (Life Technologies) and the second strand was generated using DNA Polymerase I (New England Biolabs, Ipswich, MA, USA), E. Coli DNA Ligase (New England Biolabs) and RNAse H (Life Technologies). .. Fragments between 150-300 bp were recovered after 2% agarose gel electrophoresis in order to remove adapter dimers by size selection (QIAquick Gel Extraction Kit, Qiagen).

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    New England Biolabs n dna ligase
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. 9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *

    doi: 10.1074/jbc.M115.638130

    Figure Lengend Snippet: Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. 9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.

    Article Snippet: Enzymes and Reagents All restriction endonucleases, modifying enzymes, polB (9°Nm DNA polymerase; 9°N/E143D), 9°N DNA ligase, T4 DNA ligase, nucleotides, and single-stranded M13mp18 DNA (ssM13) were from New England Biolabs, Inc. (Ipswich, MA).

    Techniques: Labeling, Blocking Assay, Incubation, Electrophoresis

    Simplified models of Okazaki fragment maturation in bacteria ( A ), Eukarya ( B ), and Thermococcus species 9°N ( C ). A, in bacteria, pol III synthesizes the lagging strand. pol I replaces pol III to complete Okazaki fragment maturation. pol I 5′-3′ exonuclease removes the RNA primer as its DNA polymerase activity fills the gap. DNA ligase seals the Okazaki fragments. B, the eukaryal lagging strand DNA polymerase, pol δ, strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. C, polD synthesizes the lagging strand and stops at a downstream Okazaki fragment. polB replaces polD and its strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. Further details are described in the text.

    Journal: The Journal of Biological Chemistry

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *

    doi: 10.1074/jbc.M115.638130

    Figure Lengend Snippet: Simplified models of Okazaki fragment maturation in bacteria ( A ), Eukarya ( B ), and Thermococcus species 9°N ( C ). A, in bacteria, pol III synthesizes the lagging strand. pol I replaces pol III to complete Okazaki fragment maturation. pol I 5′-3′ exonuclease removes the RNA primer as its DNA polymerase activity fills the gap. DNA ligase seals the Okazaki fragments. B, the eukaryal lagging strand DNA polymerase, pol δ, strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. C, polD synthesizes the lagging strand and stops at a downstream Okazaki fragment. polB replaces polD and its strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. Further details are described in the text.

    Article Snippet: Enzymes and Reagents All restriction endonucleases, modifying enzymes, polB (9°Nm DNA polymerase; 9°N/E143D), 9°N DNA ligase, T4 DNA ligase, nucleotides, and single-stranded M13mp18 DNA (ssM13) were from New England Biolabs, Inc. (Ipswich, MA).

    Techniques: Activity Assay

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Journal: The Journal of General Virology

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    doi: 10.1099/vir.0.013946-0

    Figure Lengend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Article Snippet: Restriction enzymes, reverse transcriptase SuperScript III, high-fidelity Taq DNA polymerase, Pfu polymerase, Phusion DNA polymerase, Klenow fragment of DNA polymerase, exonuclease III and DNA ligase were obtained from New England Biolabs (NEB) or Invitrogen and used according to the manufacturers' instructions.

    Techniques: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

    doi: 10.3390/ijms13033549

    Figure Lengend Snippet: In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK).

    Techniques: In Vitro, Polymerase Chain Reaction, Amplification, Purification, Marker

    An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Construct, Sequencing, Plasmid Preparation, Generated, Fluorescence

    ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Fluorescence

    DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Fluorescence, Inhibition