q5  (New England Biolabs)


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    Name:
    Q5 Site Directed Mutagenesis Kit
    Description:
    Q5 Site Directed Mutagenesis Kit 10 rxns
    Catalog Number:
    E0554S
    Price:
    197
    Category:
    PCR Mutagenesis Kits
    Size:
    10 rxns
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    Structured Review

    New England Biolabs q5
    Q5 Site Directed Mutagenesis Kit
    Q5 Site Directed Mutagenesis Kit 10 rxns
    https://www.bioz.com/result/q5/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    q5 - by Bioz Stars, 2021-09
    99/100 stars

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    Related Articles

    Generated:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Article Title: S-acylation is a positive regulator of FLS2-mediated plant immunity
    Article Snippet: .. All FLS2 mutant variants used were based on this construct and were generated using Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s guidelines. ..

    Mutagenesis:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Article Title: Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding
    Article Snippet: .. The VoC RBD mutations were introduced by site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs). ..

    Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation
    Article Snippet: .. HindIII and BamHI restriction sites were inserted just after the codon coding for proline 16 using Q5-insertion mutagenesis (#E0554; NEB) according to the protocol from the manufacturer. ..

    Article Title: Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants
    Article Snippet: .. The VoC RBD mutations were introduced by site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs). ..

    Article Title: S-acylation is a positive regulator of FLS2-mediated plant immunity
    Article Snippet: .. All FLS2 mutant variants used were based on this construct and were generated using Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s guidelines. ..

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Article Title: Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation
    Article Snippet: .. C4 domain sequence was removed with the Q5 site-directed mutagenesis kit (NEB). ..

    Article Title: Hydrogen-deuterium exchange mass spectrometry of Mtr4 with diverse RNAs reveals substrate-dependent dynamics and interfaces in the arch
    Article Snippet: .. Protein Preparation The Saccharomyces cerevisiae Mtr4ΔF was constructed by removing residues 667-813 and inserting a three-glycine linker using the Q5 Site-Directed Mutagenesis Kit (NEB). ..

    Construct:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Article Title: S-acylation is a positive regulator of FLS2-mediated plant immunity
    Article Snippet: .. All FLS2 mutant variants used were based on this construct and were generated using Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s guidelines. ..

    Article Title: Hydrogen-deuterium exchange mass spectrometry of Mtr4 with diverse RNAs reveals substrate-dependent dynamics and interfaces in the arch
    Article Snippet: .. Protein Preparation The Saccharomyces cerevisiae Mtr4ΔF was constructed by removing residues 667-813 and inserting a three-glycine linker using the Q5 Site-Directed Mutagenesis Kit (NEB). ..

    Clone Assay:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Plasmid Preparation:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Polymerase Chain Reaction:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Electroporation Bacterial Transformation:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Isolation:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Recombinant:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    DNA Sequencing:

    Article Title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration
    Article Snippet: .. Agarose gel-resolved DNAs were extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, USA); plasmid DNA and PCR amplicons were digested with commercial restriction enzymes (NEB, USA); DNAs were ligated using T4 DNA ligase (NEB, USA); competent DH5α (Invitrogen, USA) or NEB-Stbl (NEB, USA) bacterial cells were used for bacterial transformation; plasmid DNAs were isolated using the Zyppy plasmid mini prep kit or ZymoPURE II plasmid maxiprep kit (Zymo Research, USA); recombinant plasmids were verified by restriction enzyme digestion and Sanger DNA sequencing; and site-specific mutations were introduced into DNA using the Q5 Site-Directed Mutagenesis Kit (NEB, USA) and custom-made primers. ..

    Sequencing:

    Article Title: Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation
    Article Snippet: .. C4 domain sequence was removed with the Q5 site-directed mutagenesis kit (NEB). ..

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    New England Biolabs neb q5
    Macerprepped DNA is good template for PCR. 1-3, PCR product by <t>NEB</t> Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.
    Neb Q5, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neb q5/product/New England Biolabs
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    99
    New England Biolabs q5 dna polymerase
    Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) A representative quantification of dTTP using a published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31–0.08 pmol) are not shown. ( B ) A representative quantification of dTTP using <t>Q5</t> DNA polymerase, 197-nt template and EvaGreen detection chemistry. ( C , D ) dTTP signal generated by the fluorometric methods from a high concentration mouse liver extract with and without 0.5 pmol dTTP spike-in calibrant. The extract volume was adjusted to 2 μl per mg of initial tissue weight. The extract comprised half of the reaction. ( E , F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of a polynomial curve fit.
    Q5 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs atp binding site
    Overall structure and organization of the <t>GatD/MurT</t> complex. ( a ) Reaction catalyzed by GatD/MurT. The free α-carboxyl of D-iso-glutamate in the peptide stem is amidated in a glutamine- and <t>ATP-dependent</t> reaction. ( b . MurT is composed of two domains: a Mur ligase middle domain (MurT middle) containing the canonical ATP binding site and, surprisingly, a ribbon-type Zinc finger, and a C-terminal Mur ligase domain (MurT C-term). MurT residue glutamate 108 participates in ATP hydrolysis, and aspartate 349 forms the third residue in the putative catalytic triad. ( c ) Overview of the GatD/MurT structure. GatD and MurT form a boomerang-shaped complex, with GatD contacting the MurT C-term domain through contacts that are in part mediated by helix α7 of GatD. Catalytic triad residues GatD-C94, GatD-H189, MurT-D349 and the bound nucleotide AMPPNP are shown in stick representation. The zinc ion in the Cys 4 zinc ribbon of MurT is shown as a green sphere, and the four cysteine residues ligating it are shown as sticks. ( d ) Tilted view of the MurT middle domain to show the central β-sheet and the bound AMPPNP and its surrounding secondary structure elements, as well as the zinc ribbon. ( e ) Topological representation of the GatD/MurT architecture. Secondary structure nomenclature of GatD was done according to Leisico et al . As the short helices α1 and α5 in the isolated GatD structure do not conform to helical geometry in our complex, they were not assigned. The MurT domains were assigned separately with the prefixes m and c .
    Atp Binding Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp binding site/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    Image Search Results


    Macerprepped DNA is good template for PCR. 1-3, PCR product by NEB Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.

    Journal: bioRxiv

    Article Title: The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

    doi: 10.1101/2020.08.13.249607

    Figure Lengend Snippet: Macerprepped DNA is good template for PCR. 1-3, PCR product by NEB Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.

    Article Snippet: According to the NEB calculator (NEB, online tool), Tm for NEB Q5 and NEB Onetaq was determined to be 52°C and 61°C.

    Techniques: Polymerase Chain Reaction, Marker

    Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) A representative quantification of dTTP using a published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31–0.08 pmol) are not shown. ( B ) A representative quantification of dTTP using Q5 DNA polymerase, 197-nt template and EvaGreen detection chemistry. ( C , D ) dTTP signal generated by the fluorometric methods from a high concentration mouse liver extract with and without 0.5 pmol dTTP spike-in calibrant. The extract volume was adjusted to 2 μl per mg of initial tissue weight. The extract comprised half of the reaction. ( E , F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of a polynomial curve fit.

    Journal: Nucleic Acids Research

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

    doi: 10.1093/nar/gkaa516

    Figure Lengend Snippet: Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) A representative quantification of dTTP using a published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31–0.08 pmol) are not shown. ( B ) A representative quantification of dTTP using Q5 DNA polymerase, 197-nt template and EvaGreen detection chemistry. ( C , D ) dTTP signal generated by the fluorometric methods from a high concentration mouse liver extract with and without 0.5 pmol dTTP spike-in calibrant. The extract volume was adjusted to 2 μl per mg of initial tissue weight. The extract comprised half of the reaction. ( E , F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of a polynomial curve fit.

    Article Snippet: We found that the Q5 DNA polymerase, which we mainly used in this work, retains specificity in the presence of rNTP-to-dNTP ratios typically present in cultured cells.

    Techniques: Hydrolysis Assay, Generated, Concentration Assay, Fluorescence

    Optimization of EvaGreen- and Q5 DNA polymerase-based dNTP quantification. ( A–F ) Each critical reaction component and reaction temperature was varied, and the outcome evaluated. ( G ) The end-point fluorescence was measured at 66°C and after raising the temperature to 75°C to dissociate unused primers. ( H ) Assay conditions in graphs A to G. The standard curves were generated from the end-point baseline-corrected fluorescence values. The assessments were performed in triplicates. Supplementary Figure S3B shows the effect of increasing EvaGreen concentration above 1.25 μM. Supplementary Figure S3C-D shows the effect of different DNA polymerase concentrations on the dTTP and dCTP quantification.

    Journal: Nucleic Acids Research

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

    doi: 10.1093/nar/gkaa516

    Figure Lengend Snippet: Optimization of EvaGreen- and Q5 DNA polymerase-based dNTP quantification. ( A–F ) Each critical reaction component and reaction temperature was varied, and the outcome evaluated. ( G ) The end-point fluorescence was measured at 66°C and after raising the temperature to 75°C to dissociate unused primers. ( H ) Assay conditions in graphs A to G. The standard curves were generated from the end-point baseline-corrected fluorescence values. The assessments were performed in triplicates. Supplementary Figure S3B shows the effect of increasing EvaGreen concentration above 1.25 μM. Supplementary Figure S3C-D shows the effect of different DNA polymerase concentrations on the dTTP and dCTP quantification.

    Article Snippet: We found that the Q5 DNA polymerase, which we mainly used in this work, retains specificity in the presence of rNTP-to-dNTP ratios typically present in cultured cells.

    Techniques: Fluorescence, Generated, Concentration Assay

    Cloning strategy to insert HAs into the pDsRed- 24MS2 plasmid for one-step CRISPR (A) Detailed view of the target sequence that starts with a 5′G and ends with the NGG PAM site and the gRNA. HAs sit on either site of the Cas9 cleavage site, which is located in the target sequence, 3 nt away from the PAM sequence. (B) The pDsRed-24MS2 plasmid contains two MCSs for HA insertion, the DsRed marker gene flanked by loxP sites and 24xMS2 stem-loops. (C) HA1 is PCR amplified from genomic DNA (1). The plasmid backbone is linearized using restriction sites in the 5′ MCS (2) and ligated with the HA1 fragment (3). The resulting pDsRed-24MS2+HA1 plasmid is linearized by restriction digest (4) and the HA2 fragment is amplified from genomic DNA (5). HA2 is ligated into the linearized vector (6) and purified for microinjection (7). (D) The finished plasmid pDsRed-24MS2+HA1+HA2 is used as a dsDNA donor for CRISPR engineering.

    Journal: STAR Protocols

    Article Title: CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

    doi: 10.1016/j.xpro.2021.100380

    Figure Lengend Snippet: Cloning strategy to insert HAs into the pDsRed- 24MS2 plasmid for one-step CRISPR (A) Detailed view of the target sequence that starts with a 5′G and ends with the NGG PAM site and the gRNA. HAs sit on either site of the Cas9 cleavage site, which is located in the target sequence, 3 nt away from the PAM sequence. (B) The pDsRed-24MS2 plasmid contains two MCSs for HA insertion, the DsRed marker gene flanked by loxP sites and 24xMS2 stem-loops. (C) HA1 is PCR amplified from genomic DNA (1). The plasmid backbone is linearized using restriction sites in the 5′ MCS (2) and ligated with the HA1 fragment (3). The resulting pDsRed-24MS2+HA1 plasmid is linearized by restriction digest (4) and the HA2 fragment is amplified from genomic DNA (5). HA2 is ligated into the linearized vector (6) and purified for microinjection (7). (D) The finished plasmid pDsRed-24MS2+HA1+HA2 is used as a dsDNA donor for CRISPR engineering.

    Article Snippet: In short: a.Day 1: HA1 and 2 are amplified using a high-fidelity DNA polymerase and digested with restriction enzymes that recognisethe sequences that were added to the HA primers ( C, steps 1 and 5). b. HA1 is inserted into the linearized pTVCherry vector ( C, steps 2 and 3). c.Days 2–5.

    Techniques: Clone Assay, Plasmid Preparation, CRISPR, Sequencing, Marker, Polymerase Chain Reaction, Amplification, Purification

    Cloning strategy to insert HAs into the pTV cherry plasmid for two-step CRISPR (A) The pTV cherry plasmid contains two MCSs to insert HA sequences for HDR, a mini-white marker gene, loxP and FRT sites, and an attP site. It also contains a mCherry gene (not shown). (B) Detailed view of the target sequence, starting with a 5′G and ending with the NGG PAM site. The Cas9 cleavage site is located 3 nt away from the PAM sequence and the 5′ HA ends upstream of the cleavage site. (C) HA1 is amplified from genomic DNA (1), the pTV cherry plasmid is linearized using restriction sites in the 5′ MCS (2) and both are ligated (3). The pTV cherry +HA1 plasmid is linearized using restriction sites present in the 3′ MCS (4) and the HA2 insert is PCR amplified from genomic DNA (5) before it is ligated into the plasmid backbone (6). The plasmid containing both HAs is purified for microinjection (7). (D) The resulting pTV cherry +HA1+HA2 plasmid can be used as a dsDNA donor plasmid during CRISPR engineering.

    Journal: STAR Protocols

    Article Title: CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

    doi: 10.1016/j.xpro.2021.100380

    Figure Lengend Snippet: Cloning strategy to insert HAs into the pTV cherry plasmid for two-step CRISPR (A) The pTV cherry plasmid contains two MCSs to insert HA sequences for HDR, a mini-white marker gene, loxP and FRT sites, and an attP site. It also contains a mCherry gene (not shown). (B) Detailed view of the target sequence, starting with a 5′G and ending with the NGG PAM site. The Cas9 cleavage site is located 3 nt away from the PAM sequence and the 5′ HA ends upstream of the cleavage site. (C) HA1 is amplified from genomic DNA (1), the pTV cherry plasmid is linearized using restriction sites in the 5′ MCS (2) and both are ligated (3). The pTV cherry +HA1 plasmid is linearized using restriction sites present in the 3′ MCS (4) and the HA2 insert is PCR amplified from genomic DNA (5) before it is ligated into the plasmid backbone (6). The plasmid containing both HAs is purified for microinjection (7). (D) The resulting pTV cherry +HA1+HA2 plasmid can be used as a dsDNA donor plasmid during CRISPR engineering.

    Article Snippet: In short: a.Day 1: HA1 and 2 are amplified using a high-fidelity DNA polymerase and digested with restriction enzymes that recognisethe sequences that were added to the HA primers ( C, steps 1 and 5). b. HA1 is inserted into the linearized pTVCherry vector ( C, steps 2 and 3). c.Days 2–5.

    Techniques: Clone Assay, Plasmid Preparation, CRISPR, Marker, Sequencing, Amplification, Polymerase Chain Reaction, Purification

    Overall structure and organization of the GatD/MurT complex. ( a ) Reaction catalyzed by GatD/MurT. The free α-carboxyl of D-iso-glutamate in the peptide stem is amidated in a glutamine- and ATP-dependent reaction. ( b . MurT is composed of two domains: a Mur ligase middle domain (MurT middle) containing the canonical ATP binding site and, surprisingly, a ribbon-type Zinc finger, and a C-terminal Mur ligase domain (MurT C-term). MurT residue glutamate 108 participates in ATP hydrolysis, and aspartate 349 forms the third residue in the putative catalytic triad. ( c ) Overview of the GatD/MurT structure. GatD and MurT form a boomerang-shaped complex, with GatD contacting the MurT C-term domain through contacts that are in part mediated by helix α7 of GatD. Catalytic triad residues GatD-C94, GatD-H189, MurT-D349 and the bound nucleotide AMPPNP are shown in stick representation. The zinc ion in the Cys 4 zinc ribbon of MurT is shown as a green sphere, and the four cysteine residues ligating it are shown as sticks. ( d ) Tilted view of the MurT middle domain to show the central β-sheet and the bound AMPPNP and its surrounding secondary structure elements, as well as the zinc ribbon. ( e ) Topological representation of the GatD/MurT architecture. Secondary structure nomenclature of GatD was done according to Leisico et al . As the short helices α1 and α5 in the isolated GatD structure do not conform to helical geometry in our complex, they were not assigned. The MurT domains were assigned separately with the prefixes m and c .

    Journal: Scientific Reports

    Article Title: Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

    doi: 10.1038/s41598-018-31098-x

    Figure Lengend Snippet: Overall structure and organization of the GatD/MurT complex. ( a ) Reaction catalyzed by GatD/MurT. The free α-carboxyl of D-iso-glutamate in the peptide stem is amidated in a glutamine- and ATP-dependent reaction. ( b . MurT is composed of two domains: a Mur ligase middle domain (MurT middle) containing the canonical ATP binding site and, surprisingly, a ribbon-type Zinc finger, and a C-terminal Mur ligase domain (MurT C-term). MurT residue glutamate 108 participates in ATP hydrolysis, and aspartate 349 forms the third residue in the putative catalytic triad. ( c ) Overview of the GatD/MurT structure. GatD and MurT form a boomerang-shaped complex, with GatD contacting the MurT C-term domain through contacts that are in part mediated by helix α7 of GatD. Catalytic triad residues GatD-C94, GatD-H189, MurT-D349 and the bound nucleotide AMPPNP are shown in stick representation. The zinc ion in the Cys 4 zinc ribbon of MurT is shown as a green sphere, and the four cysteine residues ligating it are shown as sticks. ( d ) Tilted view of the MurT middle domain to show the central β-sheet and the bound AMPPNP and its surrounding secondary structure elements, as well as the zinc ribbon. ( e ) Topological representation of the GatD/MurT architecture. Secondary structure nomenclature of GatD was done according to Leisico et al . As the short helices α1 and α5 in the isolated GatD structure do not conform to helical geometry in our complex, they were not assigned. The MurT domains were assigned separately with the prefixes m and c .

    Article Snippet: Site-directed mutagenesis was performed according to the manufacturer´s instructions using plasmid pET21- murT/gatD as the template to generate active site mutants GatD-C94S and MurT-D349N (QuikChange Lightning Site-Directed Mutagenesis Kit, Agilent) and to generate mutations in the ATP binding site (MurT mutants T60A, E108A, N267Y and double mutant T60A E108A; Q5 site-directed mutagenesis kit, New England Biolabs).

    Techniques: Binding Assay, Isolation

    The AMPPNP binding site in GatD/MurT. ( a ) Catalytic center of MurT bound to the ATP analogue AMPPNP. The adenine base is inserted into a pocket composed of several aromatic residues and two asparagines, including N267, while the conserved K59, T60 and E108 residues coordinate the β and γ phosphates as well as a magnesium ion (green sphere) found in the active center of ATPases. A bound water is shown with a red sphere. ( b ) Superimposition of ATP analogues from S. aureus and P. aeruginosa MurF (Protein Data Bank ID 4cvk) onto the MurT ATP-binding pocket in surface representation based on structural superimpositions of the entire domains. The MurT-bound AMPPNP is shown as a colored stick model, the superimposed nucleotides from the two related structures are shown as white sticks. ( c ) Thin-layer chromatography analysis of an activity assay of ATP-binding site mutants. Mutation of the magnesium-coordinating residues T60 and E108 to alanines completely abolishes catalysis. Replacement of the conserved N267 with a bulky tyrosine residue also impedes catalysis, probably by interfering with AMPPNP binding.

    Journal: Scientific Reports

    Article Title: Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

    doi: 10.1038/s41598-018-31098-x

    Figure Lengend Snippet: The AMPPNP binding site in GatD/MurT. ( a ) Catalytic center of MurT bound to the ATP analogue AMPPNP. The adenine base is inserted into a pocket composed of several aromatic residues and two asparagines, including N267, while the conserved K59, T60 and E108 residues coordinate the β and γ phosphates as well as a magnesium ion (green sphere) found in the active center of ATPases. A bound water is shown with a red sphere. ( b ) Superimposition of ATP analogues from S. aureus and P. aeruginosa MurF (Protein Data Bank ID 4cvk) onto the MurT ATP-binding pocket in surface representation based on structural superimpositions of the entire domains. The MurT-bound AMPPNP is shown as a colored stick model, the superimposed nucleotides from the two related structures are shown as white sticks. ( c ) Thin-layer chromatography analysis of an activity assay of ATP-binding site mutants. Mutation of the magnesium-coordinating residues T60 and E108 to alanines completely abolishes catalysis. Replacement of the conserved N267 with a bulky tyrosine residue also impedes catalysis, probably by interfering with AMPPNP binding.

    Article Snippet: Site-directed mutagenesis was performed according to the manufacturer´s instructions using plasmid pET21- murT/gatD as the template to generate active site mutants GatD-C94S and MurT-D349N (QuikChange Lightning Site-Directed Mutagenesis Kit, Agilent) and to generate mutations in the ATP binding site (MurT mutants T60A, E108A, N267Y and double mutant T60A E108A; Q5 site-directed mutagenesis kit, New England Biolabs).

    Techniques: Binding Assay, Thin Layer Chromatography, Activity Assay, Mutagenesis