phusion hot start polymerase new england biolabs  (New England Biolabs)


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    Name:
    Phusion Hot Start Flex DNA Polymerase
    Description:
    Phusion Hot Start Flex DNA Polymerase 500 units
    Catalog Number:
    m0535l
    Price:
    543
    Size:
    500 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs phusion hot start polymerase new england biolabs
    Phusion Hot Start Flex DNA Polymerase
    Phusion Hot Start Flex DNA Polymerase 500 units
    https://www.bioz.com/result/phusion hot start polymerase new england biolabs/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion hot start polymerase new england biolabs - by Bioz Stars, 2020-01
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: Paragraph title: Primers, PCR conditions, and cloning of adh genes. ... PCR was performed under optimized conditions using KOD FX Neo DNA polymerase or Phusion hot start flex DNA polymerase (New England BioLabs, Tokyo, Japan) to obtain amplicons from the metagenomes.

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. The resulting amplicons were digested and cloned into the NcoI/NotI sites of pCOLADuet-1 for espO , NdeI/MfeI sites of espO/pCOLADuet-1 for espD , and NcoI/HindIII sites of pETDuet-1 for espP .

    Article Title: The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts
    Article Snippet: Paragraph title: Molecular biology and cloning ... Oligonucleotides were purchased from IDT and PCR was carried out using Phusion Hot Start Flex DNA Polymerase (New England BioLabs) or with ReddyMix PCR (Thermo Scientific).

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: All the fragments were cloned into luciferase plasmids by making the primers either containing suitable overhanging restriction enzyme sites or containing the 15-bp homologous recombination arms for SLiCE cloning ( ). .. Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Amplification:

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: The linearized vector contained 15 nucleotides at both 5′ ends for fusion with the adh gene amplified directly by PCR using metagenomic DNA. .. PCR was performed under optimized conditions using KOD FX Neo DNA polymerase or Phusion hot start flex DNA polymerase (New England BioLabs, Tokyo, Japan) to obtain amplicons from the metagenomes.

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: .. The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. PCR cycling condition – 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 10 sec, 62°C for 30 sec, 72°C for 30 sec/kb sec; 1 cycle of 72°C for 7 min; 4°C hold.

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request). .. The used set of primers provides double-coverage of the Ebola virus L (RNA-dependent RNA polymerase) gene segment to minimize primer amplification bias artifacts.

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: .. Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG. .. Thus, only fragments starting with a sequence that can be hybridized by the selective sequence of the primer were efficiently amplified.

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: .. PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C. .. The SS forward (UC74) and TM reverse (UC76) primers (5′CTCCTGCCTTATCTCRTGGCTCTGCAC 3′and 5′CACAGCCAGAGCCACYKTCCAG 3′) to amplify the signal sequence to transmembrane region (Figure ) have been described ( ).

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. Samples were amplified using a two-step protocol (98 °C, 30 sec; (98 °C, 7 sec; 72 °C, 30 sec) × 35; 72 °C, 5 min) or a touchdown PCR protocol ((98 °C, 10 s; 72–62 °C, −1 °C/cycle, 15 s; 72 °C, 30 s) × 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) × 25 cycles).

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: Paragraph title: Single-genome amplification. ... SGA PCR conditions were as follows: the first-round PCR was performed using 1 μl of cDNA dilution in a 96-well plate with 1× Phusion HF buffer, 0.2 mM each deoxynucleoside triphosphate, 0.3 μM primers SIVsm/macEnvF1 (5′-CCTCCCCCTCCAGGACTAGC-3′) and SIVsm/macEnvR1 (5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′), and 0.016 U/μl of Phusion Hot Start Flex DNA polymerase (New England BioLabs) in a 25-μl reaction volume.

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: .. We observed 100% amplification of target genes from 12 general soil metagenomic samples isolated from farms and paddy fields or garden soil samples collected in Japan; in addition, target genes from 3 genomes isolated from the genus Leifsonia ( L. poae NBRC103069, L. naganoensis NBRC103131, and L. aquatica NBRC15710) were amplified using KOD FX Neo DNA polymerase and from 8 samples of farm and bark composts in fermentation at 35 to 80°C, as well as target genes isolated from 4 genomes ( L. aurea NBRC104579, L. ginsenji NBRC104580, L. pindariensis JCM15132, and L. shinshuensis NBRC103132), were amplified using Phusion hot start flex DNA polymerase. .. Under the optimized PCR conditions, we successfully amplified genes from all 20 independent environmental metagenomes and 7 Leifsonia sp. genomes.

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: .. Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Amplicons of 200–350 bp were purified using Ampure XP beads (Beckman Coulter Genomics) according to manufacturer's instructions.

    Reporter Assay:

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: Paragraph title: Luciferase constructs and reporter assay ... Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Synthesized:

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Construct:

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. The espOD/ pCOLADuet-1 and espP /pETDuet-1 constructs were introduced into E. coli BL21(DE3) cells by electroporation.

    Article Title: The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts
    Article Snippet: Oligonucleotides were purchased from IDT and PCR was carried out using Phusion Hot Start Flex DNA Polymerase (New England BioLabs) or with ReddyMix PCR (Thermo Scientific). .. Infantis null mutants were constructed using the λ-red-recombination system and a three step PCR method to produce an amplimer containing the antibiotic resistance gene, as described in [ ].

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: Paragraph title: Luciferase constructs and reporter assay ... Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    End-sequence Profiling:

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: .. The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. PCR cycling condition – 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 10 sec, 62°C for 30 sec, 72°C for 30 sec/kb sec; 1 cycle of 72°C for 7 min; 4°C hold.

    Electrophoresis:

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. Mutation frequency was quantified using a Qiaxcel capillary electrophoresis instrument (Qiagen) as previously described .

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Adapter-ligated libraries were quantified using a Qiaxcel capillary electrophoresis instrument (Qiagen) and sequenced on an Illumina MiSeq Sequencer by the Dana-Farber Cancer Institute Molecular Biology Core.

    Incubation:

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Pre-selection libraries were also separately incubated with 2 U of BspMI restriction endonuclease (NEB) in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9) for 1 h at 37 °C. .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: Briefly, the RNA was mixed with oligo-(dT)18 primer and dNTP mixtures, heated at 65°C for 5 min, chilled on ice for 3 min, RT buffer and Maxima H Minus Enzyme Mix added, and the reaction mixture incubated at 55°C for 45 min, followed by 85°C for 45 min to inactivate the enzyme. .. PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C.

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: The reaction mixture was then incubated at 53°C for 60 min, followed by 15 min at 70°C. cDNA was stored at −80°C until further analysis. .. SGA PCR conditions were as follows: the first-round PCR was performed using 1 μl of cDNA dilution in a 96-well plate with 1× Phusion HF buffer, 0.2 mM each deoxynucleoside triphosphate, 0.3 μM primers SIVsm/macEnvF1 (5′-CCTCCCCCTCCAGGACTAGC-3′) and SIVsm/macEnvR1 (5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′), and 0.016 U/μl of Phusion Hot Start Flex DNA polymerase (New England BioLabs) in a 25-μl reaction volume.

    Luciferase:

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: Paragraph title: Luciferase constructs and reporter assay ... Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Expressing:

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Touchdown PCR:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request). .. The used set of primers provides double-coverage of the Ebola virus L (RNA-dependent RNA polymerase) gene segment to minimize primer amplification bias artifacts.

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. Samples were amplified using a two-step protocol (98 °C, 30 sec; (98 °C, 7 sec; 72 °C, 30 sec) × 35; 72 °C, 5 min) or a touchdown PCR protocol ((98 °C, 10 s; 72–62 °C, −1 °C/cycle, 15 s; 72 °C, 30 s) × 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) × 25 cycles).

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: .. Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Amplicons of 200–350 bp were purified using Ampure XP beads (Beckman Coulter Genomics) according to manufacturer's instructions.

    Modification:

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: .. Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG. .. Thus, only fragments starting with a sequence that can be hybridized by the selective sequence of the primer were efficiently amplified.

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Amplified PCR products and isolated DNA were digested with restriction endonucleases and subcloned into a modified pcDNA6 vector (Invitrogen) linearized with XhoI and SacII restriction endonuclease enzymes (NEB) and transformed into NEB Stable competent cells (NEB) following ligation reaction with T4 ligase (NEB).

    Western Blot:

    Article Title: The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts
    Article Snippet: Oligonucleotides were purchased from IDT and PCR was carried out using Phusion Hot Start Flex DNA Polymerase (New England BioLabs) or with ReddyMix PCR (Thermo Scientific). .. For western blotting, a C-terminal two-hemagglutinin (2HA) tagged version of KlfC from S .

    Transformation Assay:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: Sample preparation We analyzed seven different libraries using at least two independent sample preparations ( ): Library “PLASMID”: a pGEM3 plasmid encoding the Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga RNA-dependent RNA polymerase L, used as a surrogate system for viral nucleic acid, was transformed in MDS™42 LowMut Electrocompetent cells (Scarab genomics, Madison, WI). .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request).

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Amplified PCR products and isolated DNA were digested with restriction endonucleases and subcloned into a modified pcDNA6 vector (Invitrogen) linearized with XhoI and SacII restriction endonuclease enzymes (NEB) and transformed into NEB Stable competent cells (NEB) following ligation reaction with T4 ligase (NEB).

    Electroporation:

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. The espOD/ pCOLADuet-1 and espP /pETDuet-1 constructs were introduced into E. coli BL21(DE3) cells by electroporation.

    Transfection:

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension. .. HepG2 cells were plated one day before transfection in 96-well plates.

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: Briefly, genomic DNA was isolated 72 hours post transfection using the Agencourt DNAdvance Genomic DNA Isolation kit (Beckman Coulter Genomics) according to the manufacturer’s instructions with a Sciclone G3 liquid-handling workstation (Caliper). .. PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs).

    Ligation:

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Amplified PCR products and isolated DNA were digested with restriction endonucleases and subcloned into a modified pcDNA6 vector (Invitrogen) linearized with XhoI and SacII restriction endonuclease enzymes (NEB) and transformed into NEB Stable competent cells (NEB) following ligation reaction with T4 ligase (NEB).

    SYBR Green Assay:

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: SGA PCR conditions were as follows: the first-round PCR was performed using 1 μl of cDNA dilution in a 96-well plate with 1× Phusion HF buffer, 0.2 mM each deoxynucleoside triphosphate, 0.3 μM primers SIVsm/macEnvF1 (5′-CCTCCCCCTCCAGGACTAGC-3′) and SIVsm/macEnvR1 (5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′), and 0.016 U/μl of Phusion Hot Start Flex DNA polymerase (New England BioLabs) in a 25-μl reaction volume. .. Briefly, the melting analysis was carried out in a 96-well plate with 1.2 μl of second-round PCR product, 2 μl of IQ SYBR green supermix, and 0.16 μl of ROX passive reference dye (Bio-Rad) in an 8-μl reaction mixture using a 7500 real-time PCR system instrument (Applied Biosystems).

    Sequencing:

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: .. Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG. .. Thus, only fragments starting with a sequence that can be hybridized by the selective sequence of the primer were efficiently amplified.

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: All the resulting plasmids were verified by Sanger sequencing. .. Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C. .. The SS forward (UC74) and TM reverse (UC76) primers (5′CTCCTGCCTTATCTCRTGGCTCTGCAC 3′and 5′CACAGCCAGAGCCACYKTCCAG 3′) to amplify the signal sequence to transmembrane region (Figure ) have been described ( ).

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: .. Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Amplicons of 200–350 bp were purified using Ampure XP beads (Beckman Coulter Genomics) according to manufacturer's instructions.

    DNA Extraction:

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: Paragraph title: DNA Extraction and Modified Nextera DNA Sample Preparation ... Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG.

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: Briefly, genomic DNA was isolated 72 hours post transfection using the Agencourt DNAdvance Genomic DNA Isolation kit (Beckman Coulter Genomics) according to the manufacturer’s instructions with a Sciclone G3 liquid-handling workstation (Caliper). .. PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs).

    Marker:

    Article Title: Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing
    Article Snippet: The primers listed in and Phusion Hot Start Flex DNA polymerase (NEB) was used for all PCRs in this study; a standard PCR protocol was followed. .. The three PCR products were gel purified and used as the template in another PCR using the two flanking gene-specific primers, resulting in the Kan marker flanked by sequences adjacent to the deleted gene.

    Mutagenesis:

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: Paragraph title: Quantification of nuclease- or nickase-induced mutation rates by T7EI assay ... PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs).

    Isolation:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: Plasmid DNA was isolated with the PowerPrep HP Maxiprep System (Origene Technologies Inc., Rockville, MD) and the resulting DNA was prepared using whole shotgun procedures; Library “dsDNA”: In vitro transcribed RNAs were obtained from the pGEM3 plasmid above using T7 (positive stand) and SP6 (negative strand) RNA polymerases contained in the MAXIscript kit (Life Technologies, Grand Island, NY). .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request).

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Amplified PCR products and isolated DNA were digested with restriction endonucleases and subcloned into a modified pcDNA6 vector (Invitrogen) linearized with XhoI and SacII restriction endonuclease enzymes (NEB) and transformed into NEB Stable competent cells (NEB) following ligation reaction with T4 ligase (NEB).

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: Paragraph title: RNA Isolation, cDNA Synthesis, and PCR Amplification ... PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C.

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: Briefly, genomic DNA was isolated 72 hours post transfection using the Agencourt DNAdvance Genomic DNA Isolation kit (Beckman Coulter Genomics) according to the manufacturer’s instructions with a Sciclone G3 liquid-handling workstation (Caliper). .. PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs).

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: .. We observed 100% amplification of target genes from 12 general soil metagenomic samples isolated from farms and paddy fields or garden soil samples collected in Japan; in addition, target genes from 3 genomes isolated from the genus Leifsonia ( L. poae NBRC103069, L. naganoensis NBRC103131, and L. aquatica NBRC15710) were amplified using KOD FX Neo DNA polymerase and from 8 samples of farm and bark composts in fermentation at 35 to 80°C, as well as target genes isolated from 4 genomes ( L. aurea NBRC104579, L. ginsenji NBRC104580, L. pindariensis JCM15132, and L. shinshuensis NBRC103132), were amplified using Phusion hot start flex DNA polymerase. .. Under the optimized PCR conditions, we successfully amplified genes from all 20 independent environmental metagenomes and 7 Leifsonia sp. genomes.

    Subcloning:

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Size-exclusion Chromatography:

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. PCR cycling condition – 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 10 sec, 62°C for 30 sec, 72°C for 30 sec/kb sec; 1 cycle of 72°C for 7 min; 4°C hold.

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. Samples were amplified using a two-step protocol (98 °C, 30 sec; (98 °C, 7 sec; 72 °C, 30 sec) × 35; 72 °C, 5 min) or a touchdown PCR protocol ((98 °C, 10 s; 72–62 °C, −1 °C/cycle, 15 s; 72 °C, 30 s) × 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) × 25 cycles).

    Purification:

    Article Title: Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing
    Article Snippet: The primers listed in and Phusion Hot Start Flex DNA polymerase (NEB) was used for all PCRs in this study; a standard PCR protocol was followed. .. The three PCR products were gel purified and used as the template in another PCR using the two flanking gene-specific primers, resulting in the Kan marker flanked by sequences adjacent to the deleted gene.

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request). .. To remove fragments < 500 bp, amplicons were pooled and purified using a 0.6x AMPure XP bead size (Beckman Coulter, Indianapolis, IN).

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG. .. The dual-indexed samples were pooled and the resulting library was purified using Agencourt AMPure XP beads at 0.75 X.

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. 200 ng of purified PCR amplicons were denatured, hybridized and treated with T7 Endonuclease I (New England Biolabs).

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Amplicons of 200–350 bp were purified using Ampure XP beads (Beckman Coulter Genomics) according to manufacturer's instructions.

    Polymerase Chain Reaction:

    Article Title: YY1 regulates the germinal center reaction by inhibiting apoptosis
    Article Snippet: .. PCR to amply the YY1 locus was performed using Phusion hot start flex DNA polymerase (New England Biolabs) and primers: P1 (5’-ACCTGGTCTATCGAAAGGAAGCAC-3’), P2 (5’-GCTTCGCCTATTCCTCGCTCATAA-3’), and P4 (5’-CCAAAGTTCGAAACCTGCTTTCCT-3’) as described ( ). .. Mice were injected intraperitoneally with 1 mg BrdU.

    Article Title: Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing
    Article Snippet: .. The primers listed in and Phusion Hot Start Flex DNA polymerase (NEB) was used for all PCRs in this study; a standard PCR protocol was followed. ..

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: .. PCR was performed under optimized conditions using KOD FX Neo DNA polymerase or Phusion hot start flex DNA polymerase (New England BioLabs, Tokyo, Japan) to obtain amplicons from the metagenomes. .. The reaction conditions were based on those suggested by the manufacturer, except that the reaction mixture contained an approximately 10-fold higher concentration of each primer for KOD FX Neo DNA polymerase and 5% (vol/vol) dimethyl sulfoxide for Phusion hot start flex DNA polymerase; both polymerases offer high fidelity and robust performance.

    Article Title: Targeted metagenomics: finding rare tryptophan dimer natural products in the environment
    Article Snippet: The espO, espD, espP genes responsible for the biosynthesis of indolocarbazole core were amplified from the previously characterized esp gene cluster (NCBI accession no. ) using Phusion Hot Start Flex DNA polymerase (New England Biolabs) and primers as listed in . .. PCR cycling condition – 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 10 sec, 62°C for 30 sec, 72°C for 30 sec/kb sec; 1 cycle of 72°C for 7 min; 4°C hold.

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: The nextRAD method uses a selective PCR primer to amplify genomic loci consistently between samples. .. Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG.

    Article Title: The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts
    Article Snippet: .. Oligonucleotides were purchased from IDT and PCR was carried out using Phusion Hot Start Flex DNA Polymerase (New England BioLabs) or with ReddyMix PCR (Thermo Scientific). .. Infantis null mutants were constructed using the λ-red-recombination system and a three step PCR method to produce an amplimer containing the antibiotic resistance gene, as described in [ ].

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Amplified PCR products and isolated DNA were digested with restriction endonucleases and subcloned into a modified pcDNA6 vector (Invitrogen) linearized with XhoI and SacII restriction endonuclease enzymes (NEB) and transformed into NEB Stable competent cells (NEB) following ligation reaction with T4 ligase (NEB).

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: .. PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C. .. The SS forward (UC74) and TM reverse (UC76) primers (5′CTCCTGCCTTATCTCRTGGCTCTGCAC 3′and 5′CACAGCCAGAGCCACYKTCCAG 3′) to amplify the signal sequence to transmembrane region (Figure ) have been described ( ).

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: .. PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). .. Samples were amplified using a two-step protocol (98 °C, 30 sec; (98 °C, 7 sec; 72 °C, 30 sec) × 35; 72 °C, 5 min) or a touchdown PCR protocol ((98 °C, 10 s; 72–62 °C, −1 °C/cycle, 15 s; 72 °C, 30 s) × 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) × 25 cycles).

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: .. SGA PCR conditions were as follows: the first-round PCR was performed using 1 μl of cDNA dilution in a 96-well plate with 1× Phusion HF buffer, 0.2 mM each deoxynucleoside triphosphate, 0.3 μM primers SIVsm/macEnvF1 (5′-CCTCCCCCTCCAGGACTAGC-3′) and SIVsm/macEnvR1 (5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′), and 0.016 U/μl of Phusion Hot Start Flex DNA polymerase (New England BioLabs) in a 25-μl reaction volume. .. The second-round PCR was carried out using 1 μl of the first-round product with primers SIVmacEnvF2 (5′-TATAATAGACATGGAGACACCCTTGAGGGAGC-3′) and SIVsmEnvR2 (5′-ATGAGACATRTCTATTGCCAATTTGTA-3′) and the same PCR mixture as in the first round.

    Concentration Assay:

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: PCR was performed under optimized conditions using KOD FX Neo DNA polymerase or Phusion hot start flex DNA polymerase (New England BioLabs, Tokyo, Japan) to obtain amplicons from the metagenomes. .. The reaction conditions were based on those suggested by the manufacturer, except that the reaction mixture contained an approximately 10-fold higher concentration of each primer for KOD FX Neo DNA polymerase and 5% (vol/vol) dimethyl sulfoxide for Phusion hot start flex DNA polymerase; both polymerases offer high fidelity and robust performance.

    T7EI Assay:

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
    Article Snippet: Paragraph title: Quantification of nuclease- or nickase-induced mutation rates by T7EI assay ... PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs).

    Plasmid Preparation:

    Article Title: Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing
    Article Snippet: The primers listed in and Phusion Hot Start Flex DNA polymerase (NEB) was used for all PCRs in this study; a standard PCR protocol was followed. .. Complementary primers were used to amplify the kanamycin cassette from plasmid pBSL86 (ATCC).

    Article Title: Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
    Article Snippet: The adh gene of this vector is under the control of the tac promoter. .. PCR was performed under optimized conditions using KOD FX Neo DNA polymerase or Phusion hot start flex DNA polymerase (New England BioLabs, Tokyo, Japan) to obtain amplicons from the metagenomes.

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request). .. The used set of primers provides double-coverage of the Ebola virus L (RNA-dependent RNA polymerase) gene segment to minimize primer amplification bias artifacts.

    Article Title: The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts
    Article Snippet: Oligonucleotides were purchased from IDT and PCR was carried out using Phusion Hot Start Flex DNA Polymerase (New England BioLabs) or with ReddyMix PCR (Thermo Scientific). .. Resistant cassette was then eliminated from the genome by using a helper plasmid encoding the FLP recombinase [ ].

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: The promoterless plasmid pGL4.10 (Promega) was employed to test the putative promoter activities of FADS 1 and FADS 2. .. Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    Real-time Polymerase Chain Reaction:

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: SGA PCR conditions were as follows: the first-round PCR was performed using 1 μl of cDNA dilution in a 96-well plate with 1× Phusion HF buffer, 0.2 mM each deoxynucleoside triphosphate, 0.3 μM primers SIVsm/macEnvF1 (5′-CCTCCCCCTCCAGGACTAGC-3′) and SIVsm/macEnvR1 (5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′), and 0.016 U/μl of Phusion Hot Start Flex DNA polymerase (New England BioLabs) in a 25-μl reaction volume. .. Briefly, the melting analysis was carried out in a 96-well plate with 1.2 μl of second-round PCR product, 2 μl of IQ SYBR green supermix, and 0.16 μl of ROX passive reference dye (Bio-Rad) in an 8-μl reaction mixture using a 7500 real-time PCR system instrument (Applied Biosystems).

    RNA Extraction:

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: Roughly 1 × 106 sorted T cells were extracted for total RNA following the manufacturer’s protocol for the NucleoSpin RNA II RNA extraction kit (Machery-Nagel). .. PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C.

    Selection:

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Paragraph title: In Vitro Selection ... Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Sample Prep:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: Paragraph title: Sample preparation ... Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request).

    Article Title: Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region
    Article Snippet: Paragraph title: DNA Extraction and Modified Nextera DNA Sample Preparation ... Fragmented DNA was then amplified using Phusion Hot Start Flex DNA Polymerase (NEB), with one of the Nextera primers modified to extend 8 nucleotides into the genomic DNA with the selective sequence TGCAGGAG.

    In Vitro:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: Plasmid DNA was isolated with the PowerPrep HP Maxiprep System (Origene Technologies Inc., Rockville, MD) and the resulting DNA was prepared using whole shotgun procedures; Library “dsDNA”: In vitro transcribed RNAs were obtained from the pGEM3 plasmid above using T7 (positive stand) and SP6 (negative strand) RNA polymerases contained in the MAXIscript kit (Life Technologies, Grand Island, NY). .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request).

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Paragraph title: In Vitro Selection ... Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Next-Generation Sequencing:

    Article Title: Error baseline rates of five sample preparation methods used to characterize RNA virus populations
    Article Snippet: .. Second strand synthesis was performed using a DNA polymerase I Klenow fragment (New England Biolabs, Ipswitch, MA) and then prepared for NGS with standard whole shotgun procedures; Libraries “P_AMP” and “DS_AMP”: the pGEM3 plasmid and the cDNA obtained above were used as templates for amplicon amplification using Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA) and touchdown PCR with a panel of specific primers (sequences available upon request). .. The used set of primers provides double-coverage of the Ebola virus L (RNA-dependent RNA polymerase) gene segment to minimize primer amplification bias artifacts.

    Produced:

    Article Title: Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells
    Article Snippet: First strand cDNA was produced from 5 to 10 ng RNA following the manufacturer’s protocol for the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher). .. PCR amplification was carried out using Phusion® Hot Start Flex DNA Polymerase (NEB) in a 50 µl reaction mixture with 0.5 µl (5–10 ng) cDNA, 200 µM total dNTPs (1 µl of 10 mM stock), Phusion buffer (10 µl of 5× stock), 1 U Phusion enzyme (0.5 µl of 200 U/ml), 250 nM forward primer and 250 nM reverse primer (both 1.25 µl of 10 µM stocks), and nuclease-free water (35.5 µl), and with reaction conditions of 2 min at 98°C, 35 cycles of 98°C 10 s, 66.5°C 20 s and 72°C 60 s, and finally 10 min at 72°C.

    FLAG-tag:

    Article Title: Survey for human polyomaviruses in cancer
    Article Snippet: .. In order to generate HPyV early region expression constructs, template plasmids comprising the early regions of BKV, JCV, HPyV6, HPyV7, and HPyV10 ( ) (provided by C. Buck, National Cancer Institute, Bethesda, Maryland, USA) and KIV and WUV (Addgene, plasmid 37093 and 37094) were amplified with Phusion Hot Start Flex DNA polymerase (NEB) or Platinum Taq DNA polymerase (Invitrogen) using primer pairs comprising an N-terminal flag tag sequence, Sal I restriction endonuclease site, and a C-terminal Sac II restriction endonuclease site. .. Early regions of TSV, HPyV9, HPyV11, HPyV12, and NJPyV, with an N-terminal flag tag and flanking restriction sites for subcloning, were synthesized (GenScript , , , , and were used for the synthesis).

    High Throughput Screening Assay:

    Article Title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing
    Article Snippet: Genomic sites were amplified for deep sequencing with Phusion Hot-start FLEX DNA polymerase (NEB) and primers listed in and the touchdown PCR protocol as described above. .. Dual-indexed TruSeq Illumina deep sequencing libraries were prepared using a high-throughput “with bead” protocol (KAPA Biosystems) on a Sciclone G3 liquid-handling workstation.

    Homologous Recombination:

    Article Title: PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
    Article Snippet: All the fragments were cloned into luciferase plasmids by making the primers either containing suitable overhanging restriction enzyme sites or containing the 15-bp homologous recombination arms for SLiCE cloning ( ). .. Mutations were introduced either by site-directed mutagen with Phusion Hot Start Flex DNA Polymerase (NEB) following the manufacturer's instructions or by primer annealing and extension.

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    New England Biolabs phusion hot start flex dna polymerase
    Phusion Hot Start Flex Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion hot start flex dna polymerase/product/New England Biolabs
    Average 90 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    phusion hot start flex dna polymerase - by Bioz Stars, 2020-01
    90/100 stars
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