phusion  (New England Biolabs)


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    Structured Review

    New England Biolabs phusion
    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. <t>Phusion;</t> 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Phusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "In vitro synthesis of gene-length single-stranded DNA"

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24677-5

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Figure Legend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Techniques Used: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining

    Related Articles

    Clone Assay:

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Paragraph title: PCR Cloning, Mutagenesis, and Construction of Expression Vectors ... Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany).

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Anti-TUSC3 sera was used as a secondary screen for MagT1-null clones. .. Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs).

    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
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    Article Snippet: Standard techniques for DNA manipulation and cloning were used unless otherwise indicated ( ). .. PCR amplification was performed using Taq, Phusion or Q5 polymerase (New England Biolabs) according to the manufacturer’s recommendation.

    Amplification:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: .. Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs). .. 400 ng of PCR products were re-hybridyzed and incubated with T7E1 endonuclease (New England Biolabs) for 20 min at 37 °C.

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    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
    Article Snippet: Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease.

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
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    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
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    Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids
    Article Snippet: .. Amplification was performed with Phusion, an engineered high-fidelity DNA polymerase with enhanced processivity, using the buffers and additives supplied by the manufacturer (New England Biolabs). .. Each 20 μl reaction contained 200 μM of each dNTP, 0.5 μM of each primer, 0.2 U of enzyme and 1 ng supercoiled plasmid DNA template.

    Article Title: Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor
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    Synthesized:

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
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    Construct:

    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
    Article Snippet: Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease.

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    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
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    Electrophoresis:

    Article Title: Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells
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    Incubation:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
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    Gel Extraction:

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    Activity Assay:

    Article Title: Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon Haloferax volcanii ▿ ▿ †
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    Expressing:

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    Article Snippet: For expression of yeast proteins pRS313, pRS423, and YCpLac22 vectors were used. .. PfuII Turbo DNA Polymerase was from Stratagene and Phusion™, and High-Fidelity DNA Polymerase from New England Biolabs (Ipswich).

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Paragraph title: PCR Cloning, Mutagenesis, and Construction of Expression Vectors ... Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany).

    Article Title: CATP-6, a C. elegans Ortholog of ATP13A2 PARK9, Positively Regulates GEM-1, an SLC16A Transporter
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    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: PCR amplification is routinely used to subclone the Nb sequence into the expression vector; Nbs range from 350 to 500 bp in size. .. We routinely use Phusion or Q5 (New England Biolabs).

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: The new constructs were made by using a two-fragment PCR approach starting from the expression plasmid pTEV-16b-AtETR1 that contains the full-length Arabidopsis thaliana ethylene receptor 1 (AtETR1) cDNA. .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    Modification:

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: All truncated AtETR1 constructs and AtETR11–307 mutants were prepared in pTEV-16b vector backbone , a modified version of pET-16b (Novagen, Darmstadt, Germany) containing the N-terminal decahistidine-tag followed by a linker (SSGH) and a tobacco etch virus (TEV) protease cleavage site (ENLYFQG; instead of a Factor Xa cleavage site in pET-16b). .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    Western Blot:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Total cell extracts were prepared from colonies for protein immunoblot analysis using antibodies specific for the targeted protein (STT3A, STT3B, MagT1 or TUSC3). .. Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs).

    Transformation Assay:

    Article Title: CATP-6, a C. elegans Ortholog of ATP13A2 PARK9, Positively Regulates GEM-1, an SLC16A Transporter
    Article Snippet: For large PCR products we used either Phusion or LongAmp DNA polymerase (New England Biolabs). .. We obtained a catp-6::gfp fosmid clone derivative of WRM067B_F08 from the C. elegans TransgeneOme project and used this for transformation rescue and expression analyses.

    Derivative Assay:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Paragraph title: Generation of HEK293 derived cell lines ... Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs).

    Hybridization:

    Article Title: Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon Haloferax volcanii ▿ ▿ †
    Article Snippet: Positively charged membranes for Southern hybridization were from Ambion (Austin, TX). .. Phusion and Taq DNA polymerases, restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA).

    Southern Blot:

    Article Title: Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon Haloferax volcanii ▿ ▿ †
    Article Snippet: Phusion and Taq DNA polymerases, restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). .. Standard agarose used for the separation of DNA for Southern blotting and routine analysis was from Bio-Rad.

    Infection:

    Article Title: Aminoacyl-tRNA-Charged Eukaryotic Elongation Factor 1A Is the Bona Fide Substrate for Legionella pneumophila Effector Glucosyltransferases
    Article Snippet: Murine RAW 264.7 macrophages were used for infection studies. .. PfuII Turbo DNA Polymerase was from Stratagene and Phusion™, and High-Fidelity DNA Polymerase from New England Biolabs (Ipswich).

    Digital PCR:

    Article Title: Detection fidelity of AR mutations in plasma derived cell-free DNA
    Article Snippet: Paragraph title: Droplet digital PCR ... Genomic DNA, patient cfDNA, and water (no template control) were PCR amplified using Phusion® with Phusion® HF buffer (NEB) or Platinum SuperFi™ (Invitrogen) at loci surrounding AR amino acid 742 and AR amino acid 877/878 for either 12 or 22 cycles.

    Generated:

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs). .. Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) according to the manufacturer’s recommendations, and index codes were then added.

    DNA Sequencing:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs). .. Amplicons were cloned into pJet1.2 vector (Thermo Scientific) for DNA sequencing.

    Sequencing:

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs). .. Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) according to the manufacturer’s recommendations, and index codes were then added.

    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
    Article Snippet: Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease.

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: Paragraph title: 3.2. Amplify Nb sequence and clone into PCS2 + 8 vectors ... We routinely use Phusion or Q5 (New England Biolabs).

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment. .. The target constructs were verified by sequencing at SEQLAB Sequence Laboratories Göttingen or at the Biological-Medical Research Centre (BMFZ) of the Heinrich Heine University Düsseldorf.

    Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries
    Article Snippet: T4 DNA ligase, T4 Polynucleotide Kinase, Phusion, Proteinase K, and restriction enzymes were obtained from New England BioLabs. .. Primer synthesis and gene sequencing were performed by Integrated DNA Technologies and Genewiz, respectively.

    Imaging:

    Article Title: Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells
    Article Snippet: Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, ) and DNA was amplified (Phusion, HF kit, New England Biolabs). .. Ethidium bromide-stained DNA bands were visualized by UV imaging (MyECL imager, ThermoFisher).

    DNA Extraction:

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: The feces samples (24 in all) were snap-frozen in liquid nitrogen and then stored at -80°C until DNA extraction. .. All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs).

    Molecular Cloning:

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: Paragraph title: Molecular cloning ... Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    In Vivo:

    Article Title: CATP-6, a C. elegans Ortholog of ATP13A2 PARK9, Positively Regulates GEM-1, an SLC16A Transporter
    Article Snippet: For large PCR products we used either Phusion or LongAmp DNA polymerase (New England Biolabs). .. We obtained very similar results when we used in vivo recombination between fosmid WRM067B_F08 and a PCR fragment to generate a C-terminally tagged version of catp-6 that lacked the catp-6 3′UTR.

    Magnetic Beads:

    Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries
    Article Snippet: Sf9 cells, MyOne streptavidin magnetic beads, SybrGold, Qubit high sensitivity ds DNA quantification kit, and ethidium bromide DNA stain were purchased from Invitrogen. .. T4 DNA ligase, T4 Polynucleotide Kinase, Phusion, Proteinase K, and restriction enzymes were obtained from New England BioLabs.

    Mutagenesis:

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Paragraph title: PCR Cloning, Mutagenesis, and Construction of Expression Vectors ... Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany).

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: In short, the mutagenesis PCR primers were designed in either PCRdesign or AAscan program with a 21-nucleotides overlap for a mutagenesis primer pair. .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries
    Article Snippet: T4 DNA ligase, T4 Polynucleotide Kinase, Phusion, Proteinase K, and restriction enzymes were obtained from New England BioLabs. .. Gene mutagenesis was achieved using a QuikChange Site-Directed Mutagenesis kit.

    Isolation:

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany). .. For isolation and storage purposes, the primer pair rumClus_f/rumClus_r was used to amplify a segment of the ruminococcin-A gene cluster (GenBank accession no. ), containing rumA1A2A3 and rumM .

    Purification:

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs). .. The PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Germany).

    Polymerase Chain Reaction:

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Paragraph title: PCR Cloning, Mutagenesis, and Construction of Expression Vectors ... Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany).

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Primers for PCR amplification of genomic DNA flanking the CRISPR target sites were designed using PRIMER BLAST (NCBI). .. Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs).

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: .. All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs). .. The PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Germany).

    Article Title: CATP-6, a C. elegans Ortholog of ATP13A2 PARK9, Positively Regulates GEM-1, an SLC16A Transporter
    Article Snippet: .. For large PCR products we used either Phusion or LongAmp DNA polymerase (New England Biolabs). .. We obtained a catp-6::gfp fosmid clone derivative of WRM067B_F08 from the C. elegans TransgeneOme project and used this for transformation rescue and expression analyses.

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: PCR amplification is routinely used to subclone the Nb sequence into the expression vector; Nbs range from 350 to 500 bp in size. .. We routinely use Phusion or Q5 (New England Biolabs).

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment. .. A pair of fragments was combined into the target plasmid in Gibson assembly , as described in our earlier report .

    Article Title: Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor
    Article Snippet: .. PCR amplification was performed using Taq, Phusion or Q5 polymerase (New England Biolabs) according to the manufacturer’s recommendation. .. Genomic DNA from T. sibirica , H. denitrificans and Thioalkalivibrio sp. K90mix was prepared using the First-DNA all-tissue Kit (GEN-IAL GmbH, Troisdorf, Germany) and served as the templates in PCR reactions.

    Article Title: Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells
    Article Snippet: Paragraph title: SORBS1, 2 and 3 qRT-PCR and SORBS2 PCR for isoform identification ... Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, ) and DNA was amplified (Phusion, HF kit, New England Biolabs).

    Article Title: Detection fidelity of AR mutations in plasma derived cell-free DNA
    Article Snippet: .. Genomic DNA, patient cfDNA, and water (no template control) were PCR amplified using Phusion® with Phusion® HF buffer (NEB) or Platinum SuperFi™ (Invitrogen) at loci surrounding AR amino acid 742 and AR amino acid 877/878 for either 12 or 22 cycles. .. PCR amplification primer sequences are located in .

    Quantitative RT-PCR:

    Article Title: Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells
    Article Snippet: Paragraph title: SORBS1, 2 and 3 qRT-PCR and SORBS2 PCR for isoform identification ... Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, ) and DNA was amplified (Phusion, HF kit, New England Biolabs).

    CRISPR:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Primers for PCR amplification of genomic DNA flanking the CRISPR target sites were designed using PRIMER BLAST (NCBI). .. Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs).

    cDNA Library Assay:

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: Depending on your Nb target, template may come from a cDNA library of screened Nbs, or from commercially or publicly available plasmid sources. .. We routinely use Phusion or Q5 (New England Biolabs).

    Agarose Gel Electrophoresis:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs). .. DNA fragments were separated on a 2% agarose gel and quantified using Image Quant TL software (GE Scientific).

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: We routinely use Phusion or Q5 (New England Biolabs). .. Check for a single band by agarose gel electrophoresis, and gel extract the PCR product (Qiagen PCR and Gel clean up kits work well).

    Article Title: Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells
    Article Snippet: Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, ) and DNA was amplified (Phusion, HF kit, New England Biolabs). .. Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, ) and DNA was amplified (Phusion, HF kit, New England Biolabs).

    Polymerase Cycling Assembly:

    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
    Article Snippet: Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease.

    Plasmid Preparation:

    Article Title: Aminoacyl-tRNA-Charged Eukaryotic Elongation Factor 1A Is the Bona Fide Substrate for Legionella pneumophila Effector Glucosyltransferases
    Article Snippet: Expression
    vector pGEX-4T was from GE Healthcare (Freiburg, Germany), pET28a vector from Novagen (Madison, WI). pBC KS (+) and pBluescript KS (+) vectors were from Stratagene (Waldbronn, Germany). .. PfuII Turbo DNA Polymerase was from Stratagene and Phusion™, and High-Fidelity DNA Polymerase from New England Biolabs (Ipswich).

    Article Title: Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
    Article Snippet: Phusion® and Q5® High-Fidelity DNA polymerases were purchased from New England Biolabs (NEB) (Frankfurt am Main, Germany). .. The blunt-ended amplicons were directly inserted into a SmaI -restricted pCTUT7 vector to yield pLEO rC2 (Supplementary Figure ).

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs). .. Amplicons were cloned into pJet1.2 vector (Thermo Scientific) for DNA sequencing.

    Article Title: Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.
    Article Snippet: .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. .. Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease.

    Article Title: Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos
    Article Snippet: PCR amplification is routinely used to subclone the Nb sequence into the expression vector; Nbs range from 350 to 500 bp in size. .. We routinely use Phusion or Q5 (New England Biolabs).

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: The new constructs were made by using a two-fragment PCR approach starting from the expression plasmid pTEV-16b-AtETR1 that contains the full-length Arabidopsis thaliana ethylene receptor 1 (AtETR1) cDNA. .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids
    Article Snippet: Paragraph title: Plasmid construction ... Amplification was performed with Phusion, an engineered high-fidelity DNA polymerase with enhanced processivity, using the buffers and additives supplied by the manufacturer (New England Biolabs).

    Software:

    Article Title: Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation
    Article Snippet: Genomic fragments were amplified with Phusion or Q5 High Fidelity polymerases (New England Biolabs). .. DNA fragments were separated on a 2% agarose gel and quantified using Image Quant TL software (GE Scientific).

    Positron Emission Tomography:

    Article Title: Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1
    Article Snippet: All truncated AtETR1 constructs and AtETR11–307 mutants were prepared in pTEV-16b vector backbone , a modified version of pET-16b (Novagen, Darmstadt, Germany) containing the N-terminal decahistidine-tag followed by a linker (SSGH) and a tobacco etch virus (TEV) protease cleavage site (ENLYFQG; instead of a Factor Xa cleavage site in pET-16b). .. Each fragment was amplified in a PCR with Phusion or Q5 high-fidelity DNA polymerase (both from New England BioLabs) or purchased from Integrated DNA Technologies as a gBlocks gene fragment.

    Sample Prep:

    Article Title: The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model
    Article Snippet: All PCRs were performed using Phusion@ High-Fidelity PCR Master Mix (New England Biolabs). .. Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) according to the manufacturer’s recommendations, and index codes were then added.

    Strep-tag:

    Article Title: Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor
    Article Snippet: PCR amplification was performed using Taq, Phusion or Q5 polymerase (New England Biolabs) according to the manufacturer’s recommendation. .. The oligonucleotides used for PCR amplification and introduction of restriction sites and Strep-tag encoding sequences if necessary are listed in .

    Staining:

    Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries
    Article Snippet: Sf9 cells, MyOne streptavidin magnetic beads, SybrGold, Qubit high sensitivity ds DNA quantification kit, and ethidium bromide DNA stain were purchased from Invitrogen. .. T4 DNA ligase, T4 Polynucleotide Kinase, Phusion, Proteinase K, and restriction enzymes were obtained from New England BioLabs.

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  • 99
    New England Biolabs phusion buffer hf
    Agarose gel electrophoresis analysis of PCR fragments multiplied by <t>Phusion</t> High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.
    Phusion Buffer Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion buffer hf/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phusion buffer hf - by Bioz Stars, 2020-03
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    80
    New England Biolabs phusion hf pcr reactions
    Schematics of the subcycling <t>PCR</t> protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. <t>Phusion</t> and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
    Phusion Hf Pcr Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs phusion high fidelity dna polymerase
    The median CEL intensities for each amplicon obtained by using Stoffel <t>DNA</t> polymerase and <t>Phusion</t> DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    phusion high fidelity dna polymerase - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

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    Agarose gel electrophoresis analysis of PCR fragments multiplied by Phusion High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.

    Journal: Scientific Reports

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach

    doi: 10.1038/s41598-017-07010-4

    Figure Lengend Snippet: Agarose gel electrophoresis analysis of PCR fragments multiplied by Phusion High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.

    Article Snippet: For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis

    Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.

    Journal: PLoS ONE

    Article Title: Improved PCR Amplification of Broad Spectrum GC DNA Templates

    doi: 10.1371/journal.pone.0156478

    Figure Lengend Snippet: Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.

    Article Snippet: To address high GC content various additives were included in the Phusion HF PCR reactions as follows: 7-deaza-dGTP (NEB) at a 40:60 ratio with normal dGTP, as well as 50:50 and 60:40 ratios keeping the final concentration of dNTPs constant; DMSO (Sigma) at a final concentration of 2.5%, 5%, and 10%; betaine (Sigma) at a final concentration of 1M, 2M and 4M.

    Techniques: Polymerase Chain Reaction, Amplification, Electrophoresis, Chromatin Immunoprecipitation, Purification

    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    doi: 10.1073/pnas.0803240105

    Figure Lengend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Article Snippet: The extension was performed by addition of 0.4 units of Phusion High-Fidelity DNA Polymerase (New England Biolabs), 3 μl 1.0 mM dNTP, 5 units Ampligase (Epicenter Biotechnologies) in a 15-μl volume at 60°C for 15 min followed by 72°C for 15 min.

    Techniques: Amplification

    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Journal: Nucleic Acid Therapeutics

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    doi: 10.1089/nat.2014.0513

    Figure Lengend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification