m13k07 helper phage  (New England Biolabs)


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    Name:
    M13K07 Helper Phage
    Description:
    M13K07 Helper Phage 1 8 ml
    Catalog Number:
    n0315s
    Price:
    113
    Size:
    1 8 ml
    Category:
    Phage Display Systems Components
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    Structured Review

    New England Biolabs m13k07 helper phage
    M13K07 Helper Phage
    M13K07 Helper Phage 1 8 ml
    https://www.bioz.com/result/m13k07 helper phage/product/New England Biolabs
    Average 95 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    m13k07 helper phage - by Bioz Stars, 2021-02
    95/100 stars

    Images

    1) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    2) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    3) Product Images from "Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries"

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2012.08.008

    Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.
    Figure Legend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.

    Techniques Used: Mutagenesis, Infection, Purification

    4) Product Images from "Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy"

    Article Title: Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.11.5557-5564.2002

    Effect of conditioned medium on transduction efficiency. Actively growing E. coli (ER2738) cells in LB containing 20 μg of tetracycline/ml, in order to maintain the F′ plasmid, were briefly chilled on ice before being diluted 10,000-fold in either fresh LB containing tetracycline or filter-sterilized conditioned medium isolated from logarithmic growth or saturated cultures of the same cells. Transducing M13K07 phage carrying plasmid pBlue-GFPuv were added to the corresponding cell suspensions. Duplicate transduction mixtures were set up in parallel that contained the same number of cells and phage but in 1/10 of the volume. All mixtures were incubated at 37°C for 30 min, and then aliquots were plated on LB agar containing IPTG in triplicate. After overnight incubation, all colonies were counted and the number of transduced colonies was detected by expression of green fluorescent protein. The mean percentage of colonies transduced under each condition is shown.
    Figure Legend Snippet: Effect of conditioned medium on transduction efficiency. Actively growing E. coli (ER2738) cells in LB containing 20 μg of tetracycline/ml, in order to maintain the F′ plasmid, were briefly chilled on ice before being diluted 10,000-fold in either fresh LB containing tetracycline or filter-sterilized conditioned medium isolated from logarithmic growth or saturated cultures of the same cells. Transducing M13K07 phage carrying plasmid pBlue-GFPuv were added to the corresponding cell suspensions. Duplicate transduction mixtures were set up in parallel that contained the same number of cells and phage but in 1/10 of the volume. All mixtures were incubated at 37°C for 30 min, and then aliquots were plated on LB agar containing IPTG in triplicate. After overnight incubation, all colonies were counted and the number of transduced colonies was detected by expression of green fluorescent protein. The mean percentage of colonies transduced under each condition is shown.

    Techniques Used: Transduction, Plasmid Preparation, Isolation, Incubation, Expressing

    A fixed number of cells with serial dilutions of phage. Nine dilutions of transducing phage M13K07 lysate carrying phagemid pBlue-GFPuv were used to infect nine aliquots of 200 CFU of ER2738 host cells at a cell density of 1,000 CFU/cm 3 . After 30 min of incubation at 37°C, each reaction mixture was plated on nonselective LB agar containing IPTG and X-Gal. The percentages of blue colonies and green fluorescent protein-positive colonies were determined by direct counting and are plotted as the observed values (closed symbols). Expected values (open symbols) were calculated by finding the MOI actual for each set of reaction conditions in the experiment and multiplying the total number of colonies observed in each sample by its respective value for 1 − e −MOI actual . The results of two independent dilution series are shown. (tu, transducing units.)
    Figure Legend Snippet: A fixed number of cells with serial dilutions of phage. Nine dilutions of transducing phage M13K07 lysate carrying phagemid pBlue-GFPuv were used to infect nine aliquots of 200 CFU of ER2738 host cells at a cell density of 1,000 CFU/cm 3 . After 30 min of incubation at 37°C, each reaction mixture was plated on nonselective LB agar containing IPTG and X-Gal. The percentages of blue colonies and green fluorescent protein-positive colonies were determined by direct counting and are plotted as the observed values (closed symbols). Expected values (open symbols) were calculated by finding the MOI actual for each set of reaction conditions in the experiment and multiplying the total number of colonies observed in each sample by its respective value for 1 − e −MOI actual . The results of two independent dilution series are shown. (tu, transducing units.)

    Techniques Used: Incubation

    Phage replication at low host cell densities. Actively growing NovaBlue E. coli bacteria carrying the pBlue-GFPuv phagemid ( ) or ER2738 E. coli bacteria carrying the pBluescript phagemid ( ) were serially diluted in fresh medium and then infected with 10 10 PFU of helper phage M13K07. Aliquots were removed at 30 and 60 min postinfection, the host cells were removed by centrifugation and filtered with a 0.2-μm-pore-size filter, and in the resulting supernatants, the titers of the progeny phage that transduced ampicillin resistance and β-galactosidase expression were determined. No progeny phage were detectable at 30 min. Titers at 60 min are plotted as a function of the initial cell concentration in each culture. The slopes of the lines were plotted by linear regression and were 1.103 and 0.904, respectively. (tu, transducing units.)
    Figure Legend Snippet: Phage replication at low host cell densities. Actively growing NovaBlue E. coli bacteria carrying the pBlue-GFPuv phagemid ( ) or ER2738 E. coli bacteria carrying the pBluescript phagemid ( ) were serially diluted in fresh medium and then infected with 10 10 PFU of helper phage M13K07. Aliquots were removed at 30 and 60 min postinfection, the host cells were removed by centrifugation and filtered with a 0.2-μm-pore-size filter, and in the resulting supernatants, the titers of the progeny phage that transduced ampicillin resistance and β-galactosidase expression were determined. No progeny phage were detectable at 30 min. Titers at 60 min are plotted as a function of the initial cell concentration in each culture. The slopes of the lines were plotted by linear regression and were 1.103 and 0.904, respectively. (tu, transducing units.)

    Techniques Used: Infection, Centrifugation, Expressing, Concentration Assay

    5) Product Images from "Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage"

    Article Title: Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014702

    Tag-pVII phage display routes. ( A ) The M13K07 helper phage modified with an N -terminal pVII tag assembles into virions displaying the tag of choice on all 3–5 pVII copies on the virion tip. In principle, this modified helper phage genome is therefore analogous to a pIII phage genome vector. ( B ) Used in phagemid rescue of any standard pIII phagemid in a 3+3 system, the Tag-pVII modified helper phage yields defined, bispecific virions. Normally, such pIII phagemids render low valence display on pIII, whereas there will be multivalent Tag-pVII display. ( C ) Used in phagemid rescue of any standard pVIII phagemid in an 8+8 system, the Tag-pVII modified helper phage yields bispecific display where all pVII carry the tag, whereas the many thousand pVIII copies of the virion body will be a heterogeneous blend of POI-pVIII dispersed among wt pVIII. ( D ) When the Tag-pVII modification is integrated into a pIII phage genome display vector, a system is made in which both virion tips are fully modified with a fusion peptide on all copies of both pIII and pVII.
    Figure Legend Snippet: Tag-pVII phage display routes. ( A ) The M13K07 helper phage modified with an N -terminal pVII tag assembles into virions displaying the tag of choice on all 3–5 pVII copies on the virion tip. In principle, this modified helper phage genome is therefore analogous to a pIII phage genome vector. ( B ) Used in phagemid rescue of any standard pIII phagemid in a 3+3 system, the Tag-pVII modified helper phage yields defined, bispecific virions. Normally, such pIII phagemids render low valence display on pIII, whereas there will be multivalent Tag-pVII display. ( C ) Used in phagemid rescue of any standard pVIII phagemid in an 8+8 system, the Tag-pVII modified helper phage yields bispecific display where all pVII carry the tag, whereas the many thousand pVIII copies of the virion body will be a heterogeneous blend of POI-pVIII dispersed among wt pVIII. ( D ) When the Tag-pVII modification is integrated into a pIII phage genome display vector, a system is made in which both virion tips are fully modified with a fusion peptide on all copies of both pIII and pVII.

    Techniques Used: Modification, Plasmid Preparation

    6) Product Images from "Development of Phage Immuno-Loop-Mediated Isothermal Amplification Assays for Organophosphorus Pesticides in Agro-products"

    Article Title: Development of Phage Immuno-Loop-Mediated Isothermal Amplification Assays for Organophosphorus Pesticides in Agro-products

    Journal: Analytical Chemistry

    doi: 10.1021/ac5020657

    Sensitivity and specificity of the LAMP reaction. The sensitivity was evaluated on (A) HNB visualization of color change and (B) 2% agarose gel electrophoresis analysis of the LAMP products. Specificity was assessed by detecting 2 × 10 6 pfu mL –1 helper phage M13K07 and 1 mg μL –1 M13KE vector, the result was observed by (C) color change and (D) 2% agarose gel electrophoresis. M represented 2000 bp DNA mark; 1 represented positive control; 8 and CK represented negative control; 2 to 7 respectively represented 8.5 × 10 4 , 1.7 × 10 4 , 8.5 × 10 3 , 1.7 × 10 3 , 8.5 × 10 2 , and 1.7 × 10 2 pfu mL –1 C11-2.
    Figure Legend Snippet: Sensitivity and specificity of the LAMP reaction. The sensitivity was evaluated on (A) HNB visualization of color change and (B) 2% agarose gel electrophoresis analysis of the LAMP products. Specificity was assessed by detecting 2 × 10 6 pfu mL –1 helper phage M13K07 and 1 mg μL –1 M13KE vector, the result was observed by (C) color change and (D) 2% agarose gel electrophoresis. M represented 2000 bp DNA mark; 1 represented positive control; 8 and CK represented negative control; 2 to 7 respectively represented 8.5 × 10 4 , 1.7 × 10 4 , 8.5 × 10 3 , 1.7 × 10 3 , 8.5 × 10 2 , and 1.7 × 10 2 pfu mL –1 C11-2.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Negative Control

    7) Product Images from "Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development"

    Article Title: Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004280

    Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P
    Figure Legend Snippet: Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P

    Techniques Used: Expressing, Incubation, Fluorescence, Binding Assay, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development"

    Article Title: Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004280

    Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P
    Figure Legend Snippet: Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P

    Techniques Used: Expressing, Incubation, Fluorescence, Binding Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries"

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2012.08.008

    Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.
    Figure Legend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.

    Techniques Used: Mutagenesis, Infection, Purification

    10) Product Images from "Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development"

    Article Title: Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004280

    Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P
    Figure Legend Snippet: Buffalo portal-round three selected (Bp-R3) single-chain Fv domain (scFv) phage recognise adult Schistosoma japonicum parasites and extracts. Identical numbers of carboxyfluorescein succinimidyl ester (CFSE) conjugated non-scFv expressing M13K07 helper-phage (A1), Bp-pre (A2), Bp-R3-A (A3) or Bp-R3-ES (A4) scFv-phage pools were incubated with formaldehyde-fixed adult worm pairs. Whole S . japonicum adult parasite images captured with consistent exposure times are presented as dark field (A1, a-d) and FITC fluorescence images (A1, e-h). Three dimensional rendered image of CFSE labelled Bp-R3-AES scFv-phages binding to adult S . japonicum (B1). A total of two hundred images were captured at 10.8 μm steps with consistent exposure time. Arrows represent areas of minimal binding (B1). Cross sectional view of scFv-phage binding to adult schistosome showing binding is restricted to the surface of the parasite, with no observable binding to internal structures (B2). Bp-R3 scFv-phages bind to S . japonicum excretory-secretory (ES) protein extracts by ELISA (C). Data represent mean ± S.E.M. of the O.D. 450nm for discrete scFv-phage pools binding to ES protein preparations (C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test, where ** P

    Techniques Used: Expressing, Incubation, Fluorescence, Binding Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage"

    Article Title: Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014702

    Tag-pVII phage display routes. ( A ) The M13K07 helper phage modified with an N -terminal pVII tag assembles into virions displaying the tag of choice on all 3–5 pVII copies on the virion tip. In principle, this modified helper phage genome is therefore analogous to a pIII phage genome vector. ( B ) Used in phagemid rescue of any standard pIII phagemid in a 3+3 system, the Tag-pVII modified helper phage yields defined, bispecific virions. Normally, such pIII phagemids render low valence display on pIII, whereas there will be multivalent Tag-pVII display. ( C ) Used in phagemid rescue of any standard pVIII phagemid in an 8+8 system, the Tag-pVII modified helper phage yields bispecific display where all pVII carry the tag, whereas the many thousand pVIII copies of the virion body will be a heterogeneous blend of POI-pVIII dispersed among wt pVIII. ( D ) When the Tag-pVII modification is integrated into a pIII phage genome display vector, a system is made in which both virion tips are fully modified with a fusion peptide on all copies of both pIII and pVII.
    Figure Legend Snippet: Tag-pVII phage display routes. ( A ) The M13K07 helper phage modified with an N -terminal pVII tag assembles into virions displaying the tag of choice on all 3–5 pVII copies on the virion tip. In principle, this modified helper phage genome is therefore analogous to a pIII phage genome vector. ( B ) Used in phagemid rescue of any standard pIII phagemid in a 3+3 system, the Tag-pVII modified helper phage yields defined, bispecific virions. Normally, such pIII phagemids render low valence display on pIII, whereas there will be multivalent Tag-pVII display. ( C ) Used in phagemid rescue of any standard pVIII phagemid in an 8+8 system, the Tag-pVII modified helper phage yields bispecific display where all pVII carry the tag, whereas the many thousand pVIII copies of the virion body will be a heterogeneous blend of POI-pVIII dispersed among wt pVIII. ( D ) When the Tag-pVII modification is integrated into a pIII phage genome display vector, a system is made in which both virion tips are fully modified with a fusion peptide on all copies of both pIII and pVII.

    Techniques Used: Modification, Plasmid Preparation

    12) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    13) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    14) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    15) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    16) Product Images from "Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries"

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01759

    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
    Figure Legend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Techniques Used: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    Related Articles

    Isolation:

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries
    Article Snippet: .. Isolation and Characterization of Antigen-Specific Human VH /VL sdAbs All three VH /VL sdAb libraries were rescued with M13K07 helper phage and panned for four or five rounds against five model antigens (CEACAM6, CTLA4, GITR, HSA, and MBP-intimin). ..

    Incubation:

    Article Title: Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development
    Article Snippet: .. At OD600 = 0.5, 1 x 1011 transducing units per ml (TU/ml) M13K07 helper-phage (New England Biolabs, USA) and 25 μl 1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, USA) solution were added and culture incubated for 15 mins at 37°C without agitation. .. The culture was then centrifuged at 3500 g for 10 mins and the resulting pellet was diluted in 100 ml low-expression (LE) medium (2YT, 1% glucose, 25 mg/ml chloramphenicol (Sigma Aldrich, USA) and 0.5 mM IPTG) and shaken for 16 hr at 37°C for phage production.

    other:

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries
    Article Snippet: The cultures were superinfected with either M13K07 helper phage or M13K07ΔpIII hyper phage.

    Sequencing:

    Article Title: Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage
    Article Snippet: .. The M13K07 (New England Biolabs sequence), VCSM13 (GenBank accession no.: AY598820) and fUSE5 (GenBank accession no.: AF218364) were aligned using ClustalX 2.0.5 and manually annotated using GeneDoc ( http://www.psc.edu/biomed/genedoc ). ..

    Transformation Assay:

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries
    Article Snippet: .. With the aid of a helper virus, M13-K07, the transformed CJ236 cells secrete phage particles, from which single-stranded, circular DNA (ssDNA), containing uracil in place of thymine (i.e., uracilated), is recovered. .. A pair of mutagenic oligonucleotides are phosphorylated and annealed to the ssDNA template for in vitro synthesis of heteroduplex, double-stranded DNA (dsDNA) with T7 DNA polymerase and T4 DNA ligase.

    Binding Assay:

    Article Title: Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development
    Article Snippet: .. Signal intensity (S.I.) greater than the average of the “No DNA” negative controls plus 2.5 standard deviations (S.D.) and no binding by M13K07 helper-phage was used to determine positive recognition. ..

    Plasmid Preparation:

    Article Title: Development of Phage Immuno-Loop-Mediated Isothermal Amplification Assays for Organophosphorus Pesticides in Agro-products
    Article Snippet: .. Specificity of the LAMP was determined by performing the assay with helper phage M13K07 (2 × 106 pfu mL–1 , New England Biolabs) and M13KE vector (1 mg μL–1 , New England Biolabs). .. When the reactions were complete, the LAMP products were observed by naked eye and gel electrophoresis.

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    New England Biolabs m13k07 helper phage
    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with <t>M13K07</t> helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of
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    Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Journal: Frontiers in Immunology

    Article Title: Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

    doi: 10.3389/fimmu.2017.01759

    Figure Lengend Snippet: Impact of stability selection on human V H /V L single-domain antibody (sdAb) library sequence diversity. After transformation of Escherichia coli TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82 SS library) or protein L (VL383 SS library), then amplified by reinfection of E. coli TG1 cells. Three rounds of panning were performed, and V H /V L sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in V L (A) and V H (B) library phage and panning outputs. (C,D) Clonality of V L (C) and V H (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched V L (E) and V H (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of

    Article Snippet: The cultures were superinfected with either M13K07 helper phage or M13K07ΔpIII hyper phage.

    Techniques: Selection, Sequencing, Transformation Assay, Purification, Amplification, Next-Generation Sequencing, Functional Assay, Clone Assay

    Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.

    Journal: Methods (San Diego, Calif.)

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

    doi: 10.1016/j.ymeth.2012.08.008

    Figure Lengend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.

    Article Snippet: With the aid of a helper virus, M13-K07, the transformed CJ236 cells secrete phage particles, from which single-stranded, circular DNA (ssDNA), containing uracil in place of thymine (i.e., uracilated), is recovered.

    Techniques: Mutagenesis, Infection, Purification

    Effect of conditioned medium on transduction efficiency. Actively growing E. coli (ER2738) cells in LB containing 20 μg of tetracycline/ml, in order to maintain the F′ plasmid, were briefly chilled on ice before being diluted 10,000-fold in either fresh LB containing tetracycline or filter-sterilized conditioned medium isolated from logarithmic growth or saturated cultures of the same cells. Transducing M13K07 phage carrying plasmid pBlue-GFPuv were added to the corresponding cell suspensions. Duplicate transduction mixtures were set up in parallel that contained the same number of cells and phage but in 1/10 of the volume. All mixtures were incubated at 37°C for 30 min, and then aliquots were plated on LB agar containing IPTG in triplicate. After overnight incubation, all colonies were counted and the number of transduced colonies was detected by expression of green fluorescent protein. The mean percentage of colonies transduced under each condition is shown.

    Journal: Journal of Virology

    Article Title: Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy

    doi: 10.1128/JVI.76.11.5557-5564.2002

    Figure Lengend Snippet: Effect of conditioned medium on transduction efficiency. Actively growing E. coli (ER2738) cells in LB containing 20 μg of tetracycline/ml, in order to maintain the F′ plasmid, were briefly chilled on ice before being diluted 10,000-fold in either fresh LB containing tetracycline or filter-sterilized conditioned medium isolated from logarithmic growth or saturated cultures of the same cells. Transducing M13K07 phage carrying plasmid pBlue-GFPuv were added to the corresponding cell suspensions. Duplicate transduction mixtures were set up in parallel that contained the same number of cells and phage but in 1/10 of the volume. All mixtures were incubated at 37°C for 30 min, and then aliquots were plated on LB agar containing IPTG in triplicate. After overnight incubation, all colonies were counted and the number of transduced colonies was detected by expression of green fluorescent protein. The mean percentage of colonies transduced under each condition is shown.

    Article Snippet: Escherichia coli ER2738 and M13K07 helper phage were purchased from New England Biolabs (Beverly, Mass.).

    Techniques: Transduction, Plasmid Preparation, Isolation, Incubation, Expressing

    A fixed number of cells with serial dilutions of phage. Nine dilutions of transducing phage M13K07 lysate carrying phagemid pBlue-GFPuv were used to infect nine aliquots of 200 CFU of ER2738 host cells at a cell density of 1,000 CFU/cm 3 . After 30 min of incubation at 37°C, each reaction mixture was plated on nonselective LB agar containing IPTG and X-Gal. The percentages of blue colonies and green fluorescent protein-positive colonies were determined by direct counting and are plotted as the observed values (closed symbols). Expected values (open symbols) were calculated by finding the MOI actual for each set of reaction conditions in the experiment and multiplying the total number of colonies observed in each sample by its respective value for 1 − e −MOI actual . The results of two independent dilution series are shown. (tu, transducing units.)

    Journal: Journal of Virology

    Article Title: Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy

    doi: 10.1128/JVI.76.11.5557-5564.2002

    Figure Lengend Snippet: A fixed number of cells with serial dilutions of phage. Nine dilutions of transducing phage M13K07 lysate carrying phagemid pBlue-GFPuv were used to infect nine aliquots of 200 CFU of ER2738 host cells at a cell density of 1,000 CFU/cm 3 . After 30 min of incubation at 37°C, each reaction mixture was plated on nonselective LB agar containing IPTG and X-Gal. The percentages of blue colonies and green fluorescent protein-positive colonies were determined by direct counting and are plotted as the observed values (closed symbols). Expected values (open symbols) were calculated by finding the MOI actual for each set of reaction conditions in the experiment and multiplying the total number of colonies observed in each sample by its respective value for 1 − e −MOI actual . The results of two independent dilution series are shown. (tu, transducing units.)

    Article Snippet: Escherichia coli ER2738 and M13K07 helper phage were purchased from New England Biolabs (Beverly, Mass.).

    Techniques: Incubation

    Phage replication at low host cell densities. Actively growing NovaBlue E. coli bacteria carrying the pBlue-GFPuv phagemid ( ) or ER2738 E. coli bacteria carrying the pBluescript phagemid ( ) were serially diluted in fresh medium and then infected with 10 10 PFU of helper phage M13K07. Aliquots were removed at 30 and 60 min postinfection, the host cells were removed by centrifugation and filtered with a 0.2-μm-pore-size filter, and in the resulting supernatants, the titers of the progeny phage that transduced ampicillin resistance and β-galactosidase expression were determined. No progeny phage were detectable at 30 min. Titers at 60 min are plotted as a function of the initial cell concentration in each culture. The slopes of the lines were plotted by linear regression and were 1.103 and 0.904, respectively. (tu, transducing units.)

    Journal: Journal of Virology

    Article Title: Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy

    doi: 10.1128/JVI.76.11.5557-5564.2002

    Figure Lengend Snippet: Phage replication at low host cell densities. Actively growing NovaBlue E. coli bacteria carrying the pBlue-GFPuv phagemid ( ) or ER2738 E. coli bacteria carrying the pBluescript phagemid ( ) were serially diluted in fresh medium and then infected with 10 10 PFU of helper phage M13K07. Aliquots were removed at 30 and 60 min postinfection, the host cells were removed by centrifugation and filtered with a 0.2-μm-pore-size filter, and in the resulting supernatants, the titers of the progeny phage that transduced ampicillin resistance and β-galactosidase expression were determined. No progeny phage were detectable at 30 min. Titers at 60 min are plotted as a function of the initial cell concentration in each culture. The slopes of the lines were plotted by linear regression and were 1.103 and 0.904, respectively. (tu, transducing units.)

    Article Snippet: Escherichia coli ER2738 and M13K07 helper phage were purchased from New England Biolabs (Beverly, Mass.).

    Techniques: Infection, Centrifugation, Expressing, Concentration Assay