hifi dna assembly master mix Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs nebuilder hifi dna assembly master mix
    Further details for site-directed mutagenesis and flow cytometric screen (A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37 °C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the <t>NEBuilder</t> <t>HiFi</t> <t>DNA</t> Assembly Master Mix (1 hr, 50 °C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).
    Nebuilder Hifi Dna Assembly Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2536 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi dna assembly master mix/product/New England Biolabs
    Average 99 stars, based on 2536 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi dna assembly master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs gibson assembly
    Further details for site-directed mutagenesis and flow cytometric screen (A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37 °C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the <t>NEBuilder</t> <t>HiFi</t> <t>DNA</t> Assembly Master Mix (1 hr, 50 °C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).
    Gibson Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gibson assembly/product/New England Biolabs
    Average 99 stars, based on 2404 article reviews
    Price from $9.99 to $1999.99
    gibson assembly - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs nebuilder hifi dna assembly cloning kit
    Further details for site-directed mutagenesis and flow cytometric screen (A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37 °C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the <t>NEBuilder</t> <t>HiFi</t> <t>DNA</t> Assembly Master Mix (1 hr, 50 °C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).
    Nebuilder Hifi Dna Assembly Cloning Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi dna assembly cloning kit/product/New England Biolabs
    Average 99 stars, based on 1322 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi dna assembly cloning kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Further details for site-directed mutagenesis and flow cytometric screen (A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37 °C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the NEBuilder HiFi DNA Assembly Master Mix (1 hr, 50 °C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).

    Journal: bioRxiv

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest

    doi: 10.1101/825752

    Figure Lengend Snippet: Further details for site-directed mutagenesis and flow cytometric screen (A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37 °C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the NEBuilder HiFi DNA Assembly Master Mix (1 hr, 50 °C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).

    Article Snippet: The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Purification, Transformation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Construct, Staining, Flow Cytometry, Fluorescence

    Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Journal: PLoS ONE

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    doi: 10.1371/journal.pone.0145682

    Figure Lengend Snippet: Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Article Snippet: Depending on the total number of fragments for each assembly, certain amount of the master mixture is added to pure water to make 10 μL, to which another 10 μL of Gibson Assembly or NEBuilder HiFi DNA Assembly master mix is added.

    Techniques: Agarose Gel Electrophoresis, Staining

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction