q5 polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs q5 polymerase
    Q5 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q5 polymerase/product/New England Biolabs
    Average 99 stars, based on 170 article reviews
    Price from $9.99 to $1999.99
    q5 polymerase - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
    Article Snippet: S. cerevisiae CEN.PK2-1C strain (EUROSCARF, Frankfurt, Germany) was used as the host for homologous recombination based cloning, recombinant protein expression and surface display, and β-carotene production. .. All restriction enzymes, Q5 polymerase, and the E. coli -S. cerevisiae shuttle vectors were purchased from New England Biolabs (Ipswich, MA).

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: Molecular cloning and P. pastoris overexpression strains E. coli JM109 was used for all cloning steps. .. For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs).

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: .. Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. .. The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid isolation and the DNeasy Blood and Tissue kit (Qiagen) was used for genomic DNA isolation.

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: Genes of interest were cloned to generate fusions with YFP in either N‐ or C‐terminal orientations using the unique restriction sites of the pOpt vector concept (Lauersen et al., ). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Article Title: Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae
    Article Snippet: .. Q5 polymerase, the NEBuilderR HiFi DNA Assembly Cloning Kit, restriction enzymes, and the Quick LigationTM kit were purchased from New England Biolabs (Ipswich, MA, United States). .. For routine colony PCR, EmeraldAmp® GT PCR Master Mix was used (Clontech, Mountain View, CA, USA).

    Amplification:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: .. Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs. ..

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: .. For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: .. RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs). .. For quantitative RT–PCR analysis, 5 ng of total cDNA was subjected to real-time PCR using PowerUp SYBR Green Master Mix in the ABI 7300 Real Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: 2.3 DNA construction In order to determine in vivo protein localizations of Cr GFY1–5, the gene coding regions from start codon until the last amino acid before the stop codon of Cr GFY1–5 genes were individually PCR amplified from genomic DNA and cloned into a pOpt_mVenus_Paro backbone vector (Lauersen, Kruse, & Mussgnug, ) in‐frame with the YFP variant mVenus (hereafter YFP). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Synthesized:

    Article Title: Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae
    Article Snippet: Chemical reagents and oligonucleotides Primers were synthesized by Cosmogenetech (Seoul, Korea) and are listed in Supplementary Data . .. Q5 polymerase, the NEBuilderR HiFi DNA Assembly Cloning Kit, restriction enzymes, and the Quick LigationTM kit were purchased from New England Biolabs (Ipswich, MA, United States).

    Construct:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs. .. Purified amplicons were used to generate the final constructs using the NEBuilder HiFi DNA Assembly kit (NEB, Ipswich, MA).

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Real-time Polymerase Chain Reaction:

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs). .. For quantitative RT–PCR analysis, 5 ng of total cDNA was subjected to real-time PCR using PowerUp SYBR Green Master Mix in the ABI 7300 Real Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Incubation:

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: For anaerobic growth, nephelo flasks sealed with silicone stoppers containing 100 ml LB were flushed with argon for 45 min and needle-inoculated with 1 ml of pre-culture before incubation with weak agitation (120 rpm) to prevent precipitation. .. Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs.

    Expressing:

    Article Title: Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
    Article Snippet: S. cerevisiae CEN.PK2-1C strain (EUROSCARF, Frankfurt, Germany) was used as the host for homologous recombination based cloning, recombinant protein expression and surface display, and β-carotene production. .. All restriction enzymes, Q5 polymerase, and the E. coli -S. cerevisiae shuttle vectors were purchased from New England Biolabs (Ipswich, MA).

    Knock-In:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: All PCRs were conducted using GoTaq (Promega, M712), except for Sox2-CreERT2 5’ junction and Rosa26-FLEX 5’ junction genotyping, which were conducted using Q5 polymerase with GC enhancer due to high GC content (New England Biolabs, M0491S). .. For Sox2-P2A-mStrawberry, Sox2-P2A-CreERT2 and Rosa26-FLEX-mStrawberry knock-in experiments, primer pairs used for PCR genotyping of the 5’ and 3’ junctions consisted of one primer designed from the genomic region flanking the homology arm and another from within the inserted sequence.

    Transformation Assay:

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Over Expression:

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: .. For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Gel Purification:

    Article Title: Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes
    Article Snippet: Enzymes: Q5 Polymerase, BamHI, EcoRV, NotI, T4 ligase, and Antarctic phosphatase (New England BioLabs; NEB) and LR Clonase (Thermo Fisher Scientific). .. Omega Gel Purification Kit (Omega BioTech).

    Transfection:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs. .. The plasmids were linearized using DraIII or XmnI (NEB), transfected into endogenously tagged IMC3-3xMyc or IMC6-3xMyc RHΔhxgprt parasites, and selected for recombination at the UPRT locus using FUDR.

    Genomic Sequencing:

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4. .. Each ORF sequence was amplified with the combination of Nde I and/or Bgl II or Eco RI and/or Eco RV endonuclease restriction sites added to the 5′ of oligonucleotides to enable cloning into the pOpt vector based on the naturally occurring sites in the genomic sequences.

    Cell Culture:

    Article Title: Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes
    Article Snippet: Paragraph title: 2.1. Molecular biology and cell culture ... Enzymes: Q5 Polymerase, BamHI, EcoRV, NotI, T4 ligase, and Antarctic phosphatase (New England BioLabs; NEB) and LR Clonase (Thermo Fisher Scientific).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Paragraph title: RNA purification, RT-PCR and qRT-PCR ... RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Generated:

    Article Title: Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin
    Article Snippet: Q5 polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB). .. Site-specific mutants used in this study were generated by overlap extension PCR.

    Sequencing:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: All PCRs were conducted using GoTaq (Promega, M712), except for Sox2-CreERT2 5’ junction and Rosa26-FLEX 5’ junction genotyping, which were conducted using Q5 polymerase with GC enhancer due to high GC content (New England Biolabs, M0491S). .. For Tyr editing experiments, successful editing replaces the endogenous HinfI restriction site with an EcoRI site, while for Sox2 editing experiments, the presence of indels over the sgRNA target sequence disrupts a PciI restriction site.

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4. .. Each ORF sequence was amplified with the combination of Nde I and/or Bgl II or Eco RI and/or Eco RV endonuclease restriction sites added to the 5′ of oligonucleotides to enable cloning into the pOpt vector based on the naturally occurring sites in the genomic sequences.

    Recombinant:

    Article Title: Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
    Article Snippet: Recombinant strains were grown on synthetic complete medium consisting of 0.17% yeast nitrogen base, 0.5% ammonium sulfate, and the appropriate amino acid drop out mix, supplemented with 2% glucose (SCD). .. All restriction enzymes, Q5 polymerase, and the E. coli -S. cerevisiae shuttle vectors were purchased from New England Biolabs (Ipswich, MA).

    DNA Extraction:

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. .. The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid isolation and the DNeasy Blood and Tissue kit (Qiagen) was used for genomic DNA isolation.

    Molecular Cloning:

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: Paragraph title: Molecular cloning and P. pastoris overexpression strains ... For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs).

    In Vivo:

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: 2.3 DNA construction In order to determine in vivo protein localizations of Cr GFY1–5, the gene coding regions from start codon until the last amino acid before the stop codon of Cr GFY1–5 genes were individually PCR amplified from genomic DNA and cloned into a pOpt_mVenus_Paro backbone vector (Lauersen, Kruse, & Mussgnug, ) in‐frame with the YFP variant mVenus (hereafter YFP). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Fluorescence:

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Mutagenesis:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: Paragraph title: Plasmid construction and mutagenesis ... Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs.

    Isolation:

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. .. The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid isolation and the DNeasy Blood and Tissue kit (Qiagen) was used for genomic DNA isolation.

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Finally, total RNA was isolated using the RNeasy kit following manufacturer instructions. .. RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    Purification:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs. .. Purified amplicons were used to generate the final constructs using the NEBuilder HiFi DNA Assembly kit (NEB, Ipswich, MA).

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Paragraph title: RNA purification, RT-PCR and qRT-PCR ... RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    Article Title: Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae
    Article Snippet: For purification of fragmented DNA, we used the ExpinTM Gel SV and ExpinTM PCR SV kits. .. Q5 polymerase, the NEBuilderR HiFi DNA Assembly Cloning Kit, restriction enzymes, and the Quick LigationTM kit were purchased from New England Biolabs (Ipswich, MA, United States).

    Article Title: Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor
    Article Snippet: T4 DNA ligase, T5 exonuclease, Taq ligase, Phusion polymerase, Q5 polymerase, and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid Mini Kits were purchased from Omega Bio-tek (Norcross, GA, USA), and QIAquick PCR Purification Kits and QIAquick Gel Extraction Kits were purchased from QIAGEN (Valencia, CA, USA).

    Polymerase Chain Reaction:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: All PCRs were conducted using GoTaq (Promega, M712), except for Sox2-CreERT2 5’ junction and Rosa26-FLEX 5’ junction genotyping, which were conducted using Q5 polymerase with GC enhancer due to high GC content (New England Biolabs, M0491S). .. RFLP was performed to screen for these editing events by digesting the PCR products with EcoRI (New England Biolabs, R0101S) or PciI (New England Biolabs, R0655S), respectively, for 4 hours at 37°.

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: .. For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. .. PCR reactions were performed in an iCycler (Bio-Rad); primers used in this study are listed in Additional file .

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Contaminant DNA was removed by incubating total RNA samples with DNase (2U/μl) 37°C for at least 15 min. Any remaining DNA contamination was checked by PCR of gapdh (no visible band should be observable in RNA only samples). .. RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    Article Title: Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin
    Article Snippet: Q5 polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB). .. Site-specific mutants used in this study were generated by overlap extension PCR.

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: 2.3 DNA construction In order to determine in vivo protein localizations of Cr GFY1–5, the gene coding regions from start codon until the last amino acid before the stop codon of Cr GFY1–5 genes were individually PCR amplified from genomic DNA and cloned into a pOpt_mVenus_Paro backbone vector (Lauersen, Kruse, & Mussgnug, ) in‐frame with the YFP variant mVenus (hereafter YFP). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Article Title: Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae
    Article Snippet: For purification of fragmented DNA, we used the ExpinTM Gel SV and ExpinTM PCR SV kits. .. Q5 polymerase, the NEBuilderR HiFi DNA Assembly Cloning Kit, restriction enzymes, and the Quick LigationTM kit were purchased from New England Biolabs (Ipswich, MA, United States).

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Article Title: Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor
    Article Snippet: T4 DNA ligase, T5 exonuclease, Taq ligase, Phusion polymerase, Q5 polymerase, and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid Mini Kits were purchased from Omega Bio-tek (Norcross, GA, USA), and QIAquick PCR Purification Kits and QIAquick Gel Extraction Kits were purchased from QIAGEN (Valencia, CA, USA).

    Quantitative RT-PCR:

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Paragraph title: RNA purification, RT-PCR and qRT-PCR ... RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    Staining:

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4. .. Correct sizes of the amplifications were verified in 1.2% agarose gel stained with Midori Green (Nippongenetics).

    Nested PCR:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: For all embryo RFLP and genotyping, two nested PCR reactions were performed using an external and internal pair of primers in order to obtain enough signal from crude embryo lysate, while only the internal set of the primers were used when amplifying from mouse tail samples. .. All PCRs were conducted using GoTaq (Promega, M712), except for Sox2-CreERT2 5’ junction and Rosa26-FLEX 5’ junction genotyping, which were conducted using Q5 polymerase with GC enhancer due to high GC content (New England Biolabs, M0491S).

    Plasmid Preparation:

    Article Title: A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
    Article Snippet: Paragraph title: Plasmid construction and mutagenesis ... Coding sequences of the IMC3 and IMC6 truncations were amplified with Q5 polymerase using the online NEBuilder ( https://nebuilder.neb.com ) tool to append compatible Gibson overhangs.

    Article Title: The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris
    Article Snippet: .. For the construction of ACS1 , TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). .. Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells.

    Article Title: Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
    Article Snippet: Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. .. The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid isolation and the DNeasy Blood and Tissue kit (Qiagen) was used for genomic DNA isolation.

    Article Title: Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin
    Article Snippet: Paragraph title: DNA manipulation and plasmid construction ... Q5 polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB).

    Article Title: Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes
    Article Snippet: Genes, vectors, and DNA oligomers: Human Col1A1 and Col1A2 genes (Origene), pENTR1A vector (Thermo Fisher Scientific), pTRE-Tight vector (Clontech), pLenti.CMV/TO.DEST vectors with assorted resistance cassettes (Addgene) [ ], puromycin and hygromycin linear selection markers (Clontech), DNA primers (Sigma), and RRE, REV, and VSVG vectors for lentivirus production (Addgene). .. Enzymes: Q5 Polymerase, BamHI, EcoRV, NotI, T4 ligase, and Antarctic phosphatase (New England BioLabs; NEB) and LR Clonase (Thermo Fisher Scientific).

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: Genes of interest were cloned to generate fusions with YFP in either N‐ or C‐terminal orientations using the unique restriction sites of the pOpt vector concept (Lauersen et al., ). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Article Title: Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae
    Article Snippet: Procedures for plasmid cloning are explained in Supplementary Note . .. Q5 polymerase, the NEBuilderR HiFi DNA Assembly Cloning Kit, restriction enzymes, and the Quick LigationTM kit were purchased from New England Biolabs (Ipswich, MA, United States).

    Article Title: Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor
    Article Snippet: T4 DNA ligase, T5 exonuclease, Taq ligase, Phusion polymerase, Q5 polymerase, and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid Mini Kits were purchased from Omega Bio-tek (Norcross, GA, USA), and QIAquick PCR Purification Kits and QIAquick Gel Extraction Kits were purchased from QIAGEN (Valencia, CA, USA).

    SYBR Green Assay:

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs). .. For quantitative RT–PCR analysis, 5 ng of total cDNA was subjected to real-time PCR using PowerUp SYBR Green Master Mix in the ABI 7300 Real Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

    Selection:

    Article Title: Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes
    Article Snippet: Genes, vectors, and DNA oligomers: Human Col1A1 and Col1A2 genes (Origene), pENTR1A vector (Thermo Fisher Scientific), pTRE-Tight vector (Clontech), pLenti.CMV/TO.DEST vectors with assorted resistance cassettes (Addgene) [ ], puromycin and hygromycin linear selection markers (Clontech), DNA primers (Sigma), and RRE, REV, and VSVG vectors for lentivirus production (Addgene). .. Enzymes: Q5 Polymerase, BamHI, EcoRV, NotI, T4 ligase, and Antarctic phosphatase (New England BioLabs; NEB) and LR Clonase (Thermo Fisher Scientific).

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Agarose Gel Electrophoresis:

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4. .. Correct sizes of the amplifications were verified in 1.2% agarose gel stained with Midori Green (Nippongenetics).

    Preserving:

    Article Title: A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation
    Article Snippet: Bacterial samples were collected at different ODs, and RNALater (Thermofisher) was used to ensure RNA preservation and quality. .. RT reaction was performed using 1 μg of total RNA using SuperscriptVILO kit for 1 h. Products were amplified using either Q5 polymerase or OneTag polymerase (New England Biolabs).

    DNA Purification:

    Article Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.
    Article Snippet: Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. .. Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients.

    Lysis:

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection
    Article Snippet: Crude DNA extract was obtained from morula and blastocyst stage embryos by placing individual embryos in 10 uL of embryo lysis buffer (0.2 mg/ml Proteinase K, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20), followed by heating at 55° for 4 hours to lyse the embryos, as previously described (Chen et al., 2016). .. All PCRs were conducted using GoTaq (Promega, M712), except for Sox2-CreERT2 5’ junction and Rosa26-FLEX 5’ junction genotyping, which were conducted using Q5 polymerase with GC enhancer due to high GC content (New England Biolabs, M0491S).

    Article Title: Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin
    Article Snippet: DNA manipulation and plasmid construction S. intermedius and S. gallolyticus genomic DNA was prepared using a cell lysis buffer containing 20 mg/mL lysozyme (BioShop), 25 mM Tris–HCl (pH 8.0), and 2.5 mM EDTA, and the DNA was purified using the Genomic DNA Mini Kit (Invitrogen). .. Q5 polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB).

    Gel Extraction:

    Article Title: Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor
    Article Snippet: T4 DNA ligase, T5 exonuclease, Taq ligase, Phusion polymerase, Q5 polymerase, and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid Mini Kits were purchased from Omega Bio-tek (Norcross, GA, USA), and QIAquick PCR Purification Kits and QIAquick Gel Extraction Kits were purchased from QIAGEN (Valencia, CA, USA).

    Variant Assay:

    Article Title: Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels. Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels
    Article Snippet: 2.3 DNA construction In order to determine in vivo protein localizations of Cr GFY1–5, the gene coding regions from start codon until the last amino acid before the stop codon of Cr GFY1–5 genes were individually PCR amplified from genomic DNA and cloned into a pOpt_mVenus_Paro backbone vector (Lauersen, Kruse, & Mussgnug, ) in‐frame with the YFP variant mVenus (hereafter YFP). .. PCRs were performed using Q5 Polymerase and GC enhancer solution (New England Biolabs) following manufacturer's protocols with genomic DNA as a template; all primers used are listed in Table S4.

    Homologous Recombination:

    Article Title: Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
    Article Snippet: S. cerevisiae CEN.PK2-1C strain (EUROSCARF, Frankfurt, Germany) was used as the host for homologous recombination based cloning, recombinant protein expression and surface display, and β-carotene production. .. All restriction enzymes, Q5 polymerase, and the E. coli -S. cerevisiae shuttle vectors were purchased from New England Biolabs (Ipswich, MA).

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    New England Biolabs q5 dna polymerase
    PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for <t>Q5</t> DNA polymerase; the other three control amplicons are no larger than 3.4 kb.
    Q5 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Q5U Hot Start High Fidelity DNA Polymerase is a modified version of Q5 High Fidelity DNA Polymerase which efficiently incorporates dUTP and amplifies uracil containing DNA templates This feature is
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    PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

    Journal: Bio-protocol

    Article Title: CRISPR/Cas9 Editing of the Bacillus subtilis Genome

    doi: 10.21769/BioProtoc.2272

    Figure Lengend Snippet: PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

    Article Snippet: The PCR program used to linearize pPB41 is: PCR amplify CRISPR/Cas9 using plasmid generated in step B1 ( ) Amplify plasmid generated in step B1 (pPB43 in the example) via PCR using Q5 DNA polymerase (NEB) and primers oPEB232 and oPEB234.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Mutagenesis, Plasmid Preparation

    Assay specificity for PIK 3 CA mutations. Comparison of mutant PIK 3 CA detection in human control genomic DNA ( gDNA ) and cell‐free plasma DNA following pre‐amplification or no pre‐amplification using Q5 High‐Fidelity DNA Polymerase (New England BioLabs) or TaqMan ® PreAmp Master Mix (Life Technologies). The level of blank (LoB) was 0.04% for the mutation E545K and for H1047R, 0.02% for E542K, and less than 0.01% for H1047L as shown by the graphs.

    Journal: Molecular Oncology

    Article Title: Correlation between circulating cell‐free PIK3CA tumor DNA levels and treatment response in patients with PIK3CA‐mutated metastatic breast cancer

    doi: 10.1002/1878-0261.12305

    Figure Lengend Snippet: Assay specificity for PIK 3 CA mutations. Comparison of mutant PIK 3 CA detection in human control genomic DNA ( gDNA ) and cell‐free plasma DNA following pre‐amplification or no pre‐amplification using Q5 High‐Fidelity DNA Polymerase (New England BioLabs) or TaqMan ® PreAmp Master Mix (Life Technologies). The level of blank (LoB) was 0.04% for the mutation E545K and for H1047R, 0.02% for E542K, and less than 0.01% for H1047L as shown by the graphs.

    Article Snippet: To increase the sensitivity of the analysis, the remaining 75 μL serum DNA was subjected to 12 cycles of PCR pre‐amplification with Q5 High‐Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) using a multiplex PIK3CA primer mix.

    Techniques: Mutagenesis, Amplification

    Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

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

    Article Title: DNA synthesis from diphosphate substrates by DNA polymerases

    doi: 10.1073/pnas.1712193115

    Figure Lengend Snippet: Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

    Article Snippet: Taq DNA polymerase, Phusion High-Fidelity DNA polymerase, DeepVent DNA polymerase, Vent DNA polymerase, Q5 DNA High-Fidelity polymerase, Bst polymerase, Bsu DNA polymerase (large fragment), and ThermoPol Reaction Buffer [1×: 20 mM Tris⋅HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton–X–100, pH 8.8 at 25 °C] were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification