flid point mutants  (New England Biolabs)


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    Name:
    KLD Enzyme Mix
    Description:
    KLD Enzyme Mix 25 rxns
    Catalog Number:
    m0554s
    Price:
    312
    Size:
    25 rxns
    Category:
    PCR Mutagenesis Kits
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    Structured Review

    New England Biolabs flid point mutants
    KLD Enzyme Mix
    KLD Enzyme Mix 25 rxns
    https://www.bioz.com/result/flid point mutants/product/New England Biolabs
    Average 84 stars, based on 342 article reviews
    Price from $9.99 to $1999.99
    flid point mutants - by Bioz Stars, 2020-07
    84/100 stars

    Images

    1) Product Images from "The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation"

    Article Title: The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

    Journal: bioRxiv

    doi: 10.1101/807677

    Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.
    Figure Legend Snippet: Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.

    Techniques Used: Generated

    2) Product Images from "The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation"

    Article Title: The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

    Journal: bioRxiv

    doi: 10.1101/807677

    Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.
    Figure Legend Snippet: Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.

    Techniques Used: Generated

    3) Product Images from "The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation"

    Article Title: The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

    Journal: bioRxiv

    doi: 10.1101/807677

    Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.
    Figure Legend Snippet: Structural characterization of the native C. jejuni flagella. (a) Cryo-electron micrograph of the native C. jejuni flagella, used for the 3D reconstruction. 2D classes, generated from ∼71828 particles, are shown below. (b) EM map of the native flagellum, with helical symmetry applied, to 8.6 Å resolution. A side view is shown to the left, and top view to the right. (c) Structural model of the filament-cap complex, with FliD in surface representation and coloured as in Figure 1 , and the filament atomic model in cyan. (d) Closeup view of the various FliD-flagellin interfaces in the model shown in (c). Both termini, with the N-terminal stretch and the C-terminal helix, are shown for each FliD protomer.

    Techniques Used: Generated

    Related Articles

    Clone Assay:

    Article Title: Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2
    Article Snippet: .. Plasmids and selection phage were constructed using USER cloning or KLD Enzyme Mix (NEB, Ipswich, MA). .. DNA fragments were generated by PCR using Pfu Turbo Cx Hotstart DNA Polymerase (Agilent), VeraSeq 2.0 High Fidelity DNA Polymerase (Enzymatics), or Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific).

    Multiplex Assay:

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Amplification:

    Article Title: Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species
    Article Snippet: .. Genes of interest were PCR amplified from extracted genomic DNA, as described previously , and Gibson assembly ( ) (Gibson assembly master mix; New England BioLabs) or a KLD (kinase, ligase, DpnI) reaction (KLD Enzyme Mix, New England BioLabs) was used to insert the fragments into plasmids that had been extracted with ZymoPure midiprep and Zymo Research plasmid miniprep classic kits (Zymo Research). .. Additionally, Athe_1870 (GenBank accession number ), Calhy_0908 (GenBank accession number ), Calkr_0826 (GenBank accession number ), NA10_0869 (GenBank accession number ), and Calkro_0844 (GenBank accession number ) genes were E. coli codon optimized (without transmembrane domains or signal peptides) with the Integrated DNA Technologies (IDT) Codon Optimization Tool and synthesized by the DOE JGI on a pET-45 plasmid.

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Article Title: Targeted Intracellular Degradation of SARS-CoV-2 RBD via Computationally-Optimized Peptide Fusions
    Article Snippet: .. Single amino acid substitutions were introduced utilizing the KLD Enzyme Mix (NEB) following PCR amplification with mutagenic primers (Genewiz). .. Assembled constructs were transformed into 50 μ L NEB Turbo Competent E. coli cells, and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.

    Ligation:

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Mutagenesis:

    Article Title: Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy
    Article Snippet: .. PCR products were then blunt end ligated using KLD Enzyme Mix and KLD Reaction Buffer from the Q5 site directed mutagenesis kit (New England Biolabs, MA). .. The modified DNA constructs were used to transform Escherichia coli DH5-alpha cells, grown on kanamycin-containing agar plates and subsequently screened.

    Article Title: Enabling microbial syringol conversion through structure-guided protein engineering
    Article Snippet: .. Mutagenesis was performed using primers listed in SI Appendix , with the Q5 polymerase and KLD enzyme mix (New England BioLabs) according to the manufacturer’s protocol. .. Crystallization and Structure Determination.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

    Article Title: Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase
    Article Snippet: .. The site-directed mutagenesis PCR product was treated with KLD Enzyme Mix (NEB) before transformation into E. coli strain 5-α competent cells. .. Expression and purification of IdmH Recombinant IdmH was expressed in E. coli strain BL21(DE3) cells harbouring the pET-28_IdmH plasmid.

    Construct:

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Article Title: Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2
    Article Snippet: .. Plasmids and selection phage were constructed using USER cloning or KLD Enzyme Mix (NEB, Ipswich, MA). .. DNA fragments were generated by PCR using Pfu Turbo Cx Hotstart DNA Polymerase (Agilent), VeraSeq 2.0 High Fidelity DNA Polymerase (Enzymatics), or Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific).

    Polymerase Chain Reaction:

    Article Title: Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy
    Article Snippet: .. PCR products were then blunt end ligated using KLD Enzyme Mix and KLD Reaction Buffer from the Q5 site directed mutagenesis kit (New England Biolabs, MA). .. The modified DNA constructs were used to transform Escherichia coli DH5-alpha cells, grown on kanamycin-containing agar plates and subsequently screened.

    Article Title: Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species
    Article Snippet: .. Genes of interest were PCR amplified from extracted genomic DNA, as described previously , and Gibson assembly ( ) (Gibson assembly master mix; New England BioLabs) or a KLD (kinase, ligase, DpnI) reaction (KLD Enzyme Mix, New England BioLabs) was used to insert the fragments into plasmids that had been extracted with ZymoPure midiprep and Zymo Research plasmid miniprep classic kits (Zymo Research). .. Additionally, Athe_1870 (GenBank accession number ), Calhy_0908 (GenBank accession number ), Calkr_0826 (GenBank accession number ), NA10_0869 (GenBank accession number ), and Calkro_0844 (GenBank accession number ) genes were E. coli codon optimized (without transmembrane domains or signal peptides) with the Integrated DNA Technologies (IDT) Codon Optimization Tool and synthesized by the DOE JGI on a pET-45 plasmid.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Article Title: Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase
    Article Snippet: .. The site-directed mutagenesis PCR product was treated with KLD Enzyme Mix (NEB) before transformation into E. coli strain 5-α competent cells. .. Expression and purification of IdmH Recombinant IdmH was expressed in E. coli strain BL21(DE3) cells harbouring the pET-28_IdmH plasmid.

    Article Title: Targeted Intracellular Degradation of SARS-CoV-2 RBD via Computationally-Optimized Peptide Fusions
    Article Snippet: .. Single amino acid substitutions were introduced utilizing the KLD Enzyme Mix (NEB) following PCR amplification with mutagenic primers (Genewiz). .. Assembled constructs were transformed into 50 μ L NEB Turbo Competent E. coli cells, and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.

    Generated:

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Selection:

    Article Title: Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2
    Article Snippet: .. Plasmids and selection phage were constructed using USER cloning or KLD Enzyme Mix (NEB, Ipswich, MA). .. DNA fragments were generated by PCR using Pfu Turbo Cx Hotstart DNA Polymerase (Agilent), VeraSeq 2.0 High Fidelity DNA Polymerase (Enzymatics), or Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific).

    Sequencing:

    Article Title: Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
    Article Snippet: .. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. .. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

    Transformation Assay:

    Article Title: Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase
    Article Snippet: .. The site-directed mutagenesis PCR product was treated with KLD Enzyme Mix (NEB) before transformation into E. coli strain 5-α competent cells. .. Expression and purification of IdmH Recombinant IdmH was expressed in E. coli strain BL21(DE3) cells harbouring the pET-28_IdmH plasmid.

    Plasmid Preparation:

    Article Title: Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species
    Article Snippet: .. Genes of interest were PCR amplified from extracted genomic DNA, as described previously , and Gibson assembly ( ) (Gibson assembly master mix; New England BioLabs) or a KLD (kinase, ligase, DpnI) reaction (KLD Enzyme Mix, New England BioLabs) was used to insert the fragments into plasmids that had been extracted with ZymoPure midiprep and Zymo Research plasmid miniprep classic kits (Zymo Research). .. Additionally, Athe_1870 (GenBank accession number ), Calhy_0908 (GenBank accession number ), Calkr_0826 (GenBank accession number ), NA10_0869 (GenBank accession number ), and Calkro_0844 (GenBank accession number ) genes were E. coli codon optimized (without transmembrane domains or signal peptides) with the Integrated DNA Technologies (IDT) Codon Optimization Tool and synthesized by the DOE JGI on a pET-45 plasmid.

    Article Title: Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles
    Article Snippet: .. The previously reported d59 sortase(P94R/D160N/D165A/K190E/K196T) mutant was created by introducing the mutations via overlap extension PCR followed by ligating the linearized plasmid using Kinase–Ligase–DpnI (KLD) enzyme mix and KLD reaction buffer from the Q5 site-directed mutagenesis kit (New England Biolabs, MA, USA). .. The chemically competent E. coli DH5-α cells were transformed [Life Technologies GmbH, Frankfurt, Germany; 30 min on ice, 30 s heat shock at 42 °C followed by 37 °C for 1 h in a super optimal broth with catabolite repression medium] and plated on kanamycin-supplemented agar plates.

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    New England Biolabs kld enzyme mix
    Kld Enzyme Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kld enzyme mix/product/New England Biolabs
    Average 95 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    kld enzyme mix - by Bioz Stars, 2020-07
    95/100 stars
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