hifi dna assembly  (New England Biolabs)


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    Name:
    NEBuilder HiFi DNA Assembly Master Mix
    Description:
    NEBuilder HiFi DNA Assembly Master Mix 50 rxns
    Catalog Number:
    e2621l
    Price:
    630
    Size:
    50 rxns
    Category:
    Cloning and Expression Systems
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    Structured Review

    New England Biolabs hifi dna assembly
    NEBuilder HiFi DNA Assembly Master Mix
    NEBuilder HiFi DNA Assembly Master Mix 50 rxns
    https://www.bioz.com/result/hifi dna assembly/product/New England Biolabs
    Average 99 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    hifi dna assembly - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145682

    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.
    Figure Legend 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.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    Related Articles

    Protein Purification:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

    Generated:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

    Plasmid Preparation:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in Supplementary file 1 (PCR primers for Vif mutant library construction).

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
    Article Snippet: .. Assembly reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix. ..

    Expressing:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Polymerase Chain Reaction:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in Supplementary file 1 (PCR primers for Vif mutant library construction).

    Derivative Assay:

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction
    Article Snippet: .. We decided to investigate if Gibson Assembly kit and a recently derived kit called the NEBuilder HiFi DNA Assembly (NEB #E2621) have any inherent limitation on the number of dsDNA fragments that can be assembled at once. .. Our results prove that up to 45 dsDNA fragments can be assembled at once using these kits after we made some modifications to the standard protocols to limit the 5’ exonuclease activity on shorter dsDNA fragments.

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  • 99
    New England Biolabs flag atrxδrbr ha
    RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and <t>ATRXΔRBR</t> (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with <t>FLAG-ATRX</t> helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.
    Flag Atrxδrbr Ha, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag atrxδrbr ha/product/New England Biolabs
    Average 99 stars, based on 130 article reviews
    Price from $9.99 to $1999.99
    flag atrxδrbr ha - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and ATRXΔRBR (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with FLAG-ATRX helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function

    doi: 10.1038/s41467-020-15902-9

    Figure Lengend Snippet: RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and ATRXΔRBR (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with FLAG-ATRX helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.

    Article Snippet: Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S).

    Techniques: Sequencing, RNA Binding Assay, Purification, In Vitro, Staining, Immunoprecipitation, Western Blot

    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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

    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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

    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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

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

    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