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  • 90
    TaKaRa in fusion hd cloning kit
    Overview of <t>In-Fusion</t> enzyme-based <t>cloning</t> of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® <t>HD</t> cloning <t>kit</t> with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
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    TaKaRa in fusion hd cloning system
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion ecodry hd cloning kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Ecodry Hd Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd cloning plus kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning Plus Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd ecodry cloning system
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Ecodry Cloning System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd ecodry cloning plus
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Ecodry Cloning Plus, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd ecodry cloning kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Ecodry Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa online in fusion hd cloning kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    Online In Fusion Hd Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd cloning plus ce kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning Plus Ce Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd cloning system ce kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning System Ce Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa technology kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    Technology Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa stellar competent cells
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    Stellar Competent Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1026 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa system kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    System Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 83/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa infusion hd kits
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    Infusion Hd Kits, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

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

    Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, Construct

    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent cloning. HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption in Fusarium fujikuroi created using the In-Fusion cloning system. NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.

    Journal: The Plant Pathology Journal

    Article Title: Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    doi: 10.5423/PPJ.OA.12.2015.0263

    Figure Lengend Snippet: Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent cloning. HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption in Fusarium fujikuroi created using the In-Fusion cloning system. NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.

    Article Snippet: Primer design for Fusarium cyclin C1 gene (FCC1 ) and FUM1 deletions To generate target gene deletion mutants using the In-Fusion® HD Cloning system (Clontech Laboratories Inc., Mountain View, CA, USA), primers were designed using a primer design tool developed by In-Fusion® HD Cloning.

    Techniques: Clone Assay, Construct

    Fusarium cyclin C1 ( FCC1 ) disruption in the Fusarium fujikuroi strain B14 using the In-Fusion cloning system. (A) Strategies for FCC1 disruption by homologous recombination. (B) Amplification of the three primary products: the 5′ flanking region of FCC1 , neomycin phosphotransferase ( NPTII ), and the 3′ flanking region of FCC1 . (C) Verification of the constructs prior to target gene disruption. (D) PCR screening results from the selected transformants using specific primers. P, positive control. (E) Southern blot analysis of wild-type and fcc1 mutants.

    Journal: The Plant Pathology Journal

    Article Title: Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    doi: 10.5423/PPJ.OA.12.2015.0263

    Figure Lengend Snippet: Fusarium cyclin C1 ( FCC1 ) disruption in the Fusarium fujikuroi strain B14 using the In-Fusion cloning system. (A) Strategies for FCC1 disruption by homologous recombination. (B) Amplification of the three primary products: the 5′ flanking region of FCC1 , neomycin phosphotransferase ( NPTII ), and the 3′ flanking region of FCC1 . (C) Verification of the constructs prior to target gene disruption. (D) PCR screening results from the selected transformants using specific primers. P, positive control. (E) Southern blot analysis of wild-type and fcc1 mutants.

    Article Snippet: Primer design for Fusarium cyclin C1 gene (FCC1 ) and FUM1 deletions To generate target gene deletion mutants using the In-Fusion® HD Cloning system (Clontech Laboratories Inc., Mountain View, CA, USA), primers were designed using a primer design tool developed by In-Fusion® HD Cloning.

    Techniques: Clone Assay, Homologous Recombination, Amplification, Construct, Polymerase Chain Reaction, Positive Control, Southern Blot