in fusion hd cloning kit clontech takara  (TaKaRa)

 
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    Name:
    In Fusion HD Cloning Kit
    Description:
    In Fusion Cloning offers a one step high efficiency and high throughput cloning method for projects that require a large number of cloning reactions such as expression screening studies and library construction
    Catalog Number:
    639650
    Price:
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    Size:
    100 Rxns
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    In Fusion Cloning for high throughput workflows In Fusion Cloning Cloning
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    Structured Review

    TaKaRa in fusion hd cloning kit clontech takara
    In Fusion Cloning offers a one step high efficiency and high throughput cloning method for projects that require a large number of cloning reactions such as expression screening studies and library construction
    https://www.bioz.com/result/in fusion hd cloning kit clontech takara/product/TaKaRa
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning kit clontech takara - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
    Article Snippet: .. The Citrine responder (pJFRC81_3×VAS1 -Syn21-Citrine-HA-P10) was assembled from plasmid pJFRC81_3×VAS-1-GFP-P10 digested with BglII and EcoRI, and fragments Syn21-Citrine-HA, VAS-1-HA-P10, Mhc-SD by in-fusion cloning (Clontech, 639648). .. The Cerulean responder was assembled from plasmid 3×VAS-2-GFP-P10 digested with AatII and EcoRI, and fragments Hsp70-Syn21, FLAG-Cerulean and VAS-2-P10 by in-fusion cloning.

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories). .. The resulting plasmid DNA, designated p(pulB), was used to transform Competent High DH5α (Toyobo, Tokyo, Japan) cells according to the manufacturer's instructions.

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech) .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare)

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: .. The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan). .. To yield the p35S–HHm-SYNV MRGFP-RFP , the HH2 catalytic sites were mutated from GUC to GUG by mutagenesis PCR using the primers TG+ SYNV(+)1 Xma I F and T-DNA LB Spe I Pvu I R, the resulting HHm PCR products were digested with Xma I and Sac I and then inserted into the intermediate plasmid.

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: .. The amplified pulB gene was ligated into the pCold™ TF DNA plasmid using the In-Fusion HD Cloning Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s instructions. .. The resulting plasmid DNA was used to transform Competent High DH5α (Toyobo, Tokyo, Japan) according to the manufacturer's instructions, and the transformed E . coli cells were subsequently spread onto LB agar plates containing 50 μg/ml ampicillin.

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
    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. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
    Article Snippet: .. The mCherry responder was assembled from plasmid pJFRC81_3×VAS-3-GFP-P10 (digested with BglII and EcoRI) and fragments Syn21-V5-mCherry, VAS-3-P10, Mhc-SA by in-fusion cloning (Clontech, 639648) resulting in pJFRC81_3×VAS3 -Syn21-V5-mCherry-P10. .. For the generation of a Citrine-Cerulean double responder vector, the 3×VAS4 -hsp70-Syn21-FLAG-Cerulean-P10 cassette was amplified from plasmid pJFRC81_3×VAS4 -Syn21-FLAG-Cerulaen-P10 using primers 3 ×VAS 4 -Cerulean-Fwd and 3 ×VAS 4 -Cerulean-Rev .

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: .. Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech). .. The correct sequences of wild-type (WT; pTB2ex13WT ) and mutant plasmids (pTB2ex13DEL ) were verified by DNA sequencing.

    Amplification:

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: .. The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan). .. To yield the p35S–HHm-SYNV MRGFP-RFP , the HH2 catalytic sites were mutated from GUC to GUG by mutagenesis PCR using the primers TG+ SYNV(+)1 Xma I F and T-DNA LB Spe I Pvu I R, the resulting HHm PCR products were digested with Xma I and Sac I and then inserted into the intermediate plasmid.

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: .. The amplified pulB gene was ligated into the pCold™ TF DNA plasmid using the In-Fusion HD Cloning Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s instructions. .. The resulting plasmid DNA was used to transform Competent High DH5α (Toyobo, Tokyo, Japan) according to the manufacturer's instructions, and the transformed E . coli cells were subsequently spread onto LB agar plates containing 50 μg/ml ampicillin.

    Purification:

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
    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. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

    Polymerase Chain Reaction:

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: .. The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan). .. To yield the p35S–HHm-SYNV MRGFP-RFP , the HH2 catalytic sites were mutated from GUC to GUG by mutagenesis PCR using the primers TG+ SYNV(+)1 Xma I F and T-DNA LB Spe I Pvu I R, the resulting HHm PCR products were digested with Xma I and Sac I and then inserted into the intermediate plasmid.

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: .. Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech). .. The correct sequences of wild-type (WT; pTB2ex13WT ) and mutant plasmids (pTB2ex13DEL ) were verified by DNA sequencing.

    Gel Extraction:

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
    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. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
    Article Snippet: .. Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

    Over Expression:

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech) .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare)

    Plasmid Preparation:

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
    Article Snippet: .. The Citrine responder (pJFRC81_3×VAS1 -Syn21-Citrine-HA-P10) was assembled from plasmid pJFRC81_3×VAS-1-GFP-P10 digested with BglII and EcoRI, and fragments Syn21-Citrine-HA, VAS-1-HA-P10, Mhc-SD by in-fusion cloning (Clontech, 639648). .. The Cerulean responder was assembled from plasmid 3×VAS-2-GFP-P10 digested with AatII and EcoRI, and fragments Hsp70-Syn21, FLAG-Cerulean and VAS-2-P10 by in-fusion cloning.

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech) .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare)

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: .. The amplified pulB gene was ligated into the pCold™ TF DNA plasmid using the In-Fusion HD Cloning Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s instructions. .. The resulting plasmid DNA was used to transform Competent High DH5α (Toyobo, Tokyo, Japan) according to the manufacturer's instructions, and the transformed E . coli cells were subsequently spread onto LB agar plates containing 50 μg/ml ampicillin.

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
    Article Snippet: .. The mCherry responder was assembled from plasmid pJFRC81_3×VAS-3-GFP-P10 (digested with BglII and EcoRI) and fragments Syn21-V5-mCherry, VAS-3-P10, Mhc-SA by in-fusion cloning (Clontech, 639648) resulting in pJFRC81_3×VAS3 -Syn21-V5-mCherry-P10. .. For the generation of a Citrine-Cerulean double responder vector, the 3×VAS4 -hsp70-Syn21-FLAG-Cerulean-P10 cassette was amplified from plasmid pJFRC81_3×VAS4 -Syn21-FLAG-Cerulaen-P10 using primers 3 ×VAS 4 -Cerulean-Fwd and 3 ×VAS 4 -Cerulean-Rev .

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: .. Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech). .. The correct sequences of wild-type (WT; pTB2ex13WT ) and mutant plasmids (pTB2ex13DEL ) were verified by DNA sequencing.

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    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
    In Fusion Hd Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in fusion hd cloning kit/product/TaKaRa
    Average 99 stars, based on 2240 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

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    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

    Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, Quantitative RT-PCR, In Vivo, Produced, Reverse Transcription Polymerase Chain Reaction, Amplification, Activity Assay, Construct, Fluorescence, Microscopy, Western Blot

    Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Microscopy, Western Blot, Staining, Northern Blot, Quantitative RT-PCR, Isolation

    Substitutions in Poly(T) Tail Promote Inclusion of the AluY Exon (A) Schematic representation of the minigene including AluY exon (gray square), exon 13 (white square), intronic region (black lines). The wild-type sequence (pTB2ex13 WT ) as well as the mutated sequence (pTB2ex13 WT_5mut ) are depicted below. Point variations are highlighted in gray. The AluY exonized sequence is underlined. (B and C) RT-PCR results obtained after transfection of HeLa (B) or Huh7 (C) cells with pTB2ex13 WT (lines 1 to 3) and pTB2ex13 WT_5mut (lines 4 to 6). Abbreviations: Ex, exon; M, marker size; bp, base pair.

    Journal: American Journal of Human Genetics

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization

    doi: 10.1016/j.ajhg.2017.12.010

    Figure Lengend Snippet: Substitutions in Poly(T) Tail Promote Inclusion of the AluY Exon (A) Schematic representation of the minigene including AluY exon (gray square), exon 13 (white square), intronic region (black lines). The wild-type sequence (pTB2ex13 WT ) as well as the mutated sequence (pTB2ex13 WT_5mut ) are depicted below. Point variations are highlighted in gray. The AluY exonized sequence is underlined. (B and C) RT-PCR results obtained after transfection of HeLa (B) or Huh7 (C) cells with pTB2ex13 WT (lines 1 to 3) and pTB2ex13 WT_5mut (lines 4 to 6). Abbreviations: Ex, exon; M, marker size; bp, base pair.

    Article Snippet: Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech).

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Marker

    Comparison of phenotypes and genotypes between parental ey Δ877/Δ1 strain and HR revertants. ( a ) Lateral pictures of wild type (left), parental ey Δ877/Δ1 strain (centre), and HR revertant (right). The ey Δ877/Δ1 strain shows the deformed, non-spherical compound eye (ce) phenotype. The HR revertant shows the normal eye phenotype and wild type strain. Scale bar indicates 100 μm. ( b ) Schematic illustration of genomic PCR to check the integration of attP into the Dma-ey Δ1 allele. Black thick bar indicates the Dma-ey Δ1 allele. Primers are shown by arrows. The attP knock-in results in 240 bp long PCR products, whereas no modification gives 173 bp long PCR products. ( c ) Agarose gel electrophoresis of genomic PCR products using HR revertant strains. 1 and 2 are derived from co-injection of TALEN mRNAs and targeting plasmid, whereas 3 is derived from co-injection of TALEN mRNAs and ssODN with 80 nt homology. attP knocked-in HR revertants showed 240 bp PCR products, whereas parental ey Δ877/Δ1 strain showed 173 bp PCR products. M indicates the marker DNA.

    Journal: Scientific Reports

    Article Title: TALEN-mediated homologous recombination in Daphnia magna

    doi: 10.1038/srep18312

    Figure Lengend Snippet: Comparison of phenotypes and genotypes between parental ey Δ877/Δ1 strain and HR revertants. ( a ) Lateral pictures of wild type (left), parental ey Δ877/Δ1 strain (centre), and HR revertant (right). The ey Δ877/Δ1 strain shows the deformed, non-spherical compound eye (ce) phenotype. The HR revertant shows the normal eye phenotype and wild type strain. Scale bar indicates 100 μm. ( b ) Schematic illustration of genomic PCR to check the integration of attP into the Dma-ey Δ1 allele. Black thick bar indicates the Dma-ey Δ1 allele. Primers are shown by arrows. The attP knock-in results in 240 bp long PCR products, whereas no modification gives 173 bp long PCR products. ( c ) Agarose gel electrophoresis of genomic PCR products using HR revertant strains. 1 and 2 are derived from co-injection of TALEN mRNAs and targeting plasmid, whereas 3 is derived from co-injection of TALEN mRNAs and ssODN with 80 nt homology. attP knocked-in HR revertants showed 240 bp PCR products, whereas parental ey Δ877/Δ1 strain showed 173 bp PCR products. M indicates the marker DNA.

    Article Snippet: Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA).

    Techniques: Polymerase Chain Reaction, Knock-In, Modification, Agarose Gel Electrophoresis, Derivative Assay, Injection, Plasmid Preparation, Marker

    Establishment and use of Dma-lig4 deficient mutant by CRISPR/Cas. ( a ) Schematic gene structure of Dma-lig4 . Dma-lig4 gene is annotated to consist of 8 exons (open boxes). Putative catalytic domain-encoding regions are highlighted in yellow (adenylation domain) and light green (oligonucleotide binding fold domain). gRNA-targeted sites are shown by triangles. Primers used for RT-PCR are shown by black arrows. Scale bar indicates 0.2 kb. ( b ) PAGE analyses around gRNA #2 target site of putative Dma-lig4 mutants. The genomic region surrounding gRNA #2 target site was amplified from genome DNAs of randomly selected 18 G1 lines. The original size of the PCR products should be 145 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formation on the gRNA #2 targeted site. Asterisks (*) show the putative monoallelic and/or biallelic mutants. M indicates the marker DNA. ( c ) Genotype of Dma-lig4 biallelic mutant strain. Deduced amino acid sequences of wild type or Dma-lig4 mutant #14 are shown under the genome DNA sequences. gRNA #2 targeted sequences and Protospacer Adjacent Motif (PAM) are lined. Especially, gRNA #2 targeted sequences are highlighted in bold. Lower case indicates the introduced in-del mutations into mutant #14 genome. As shown in red, a premature termination codon was introduced into both Dma-lig4 alleles in the mutant #14 genome. ( d , e ) PAGE analyses of G1 offspring from TALEN and targeting plasmid injection using ey Δ877/Δ1 strain with or without Dma-ey deficiency (d, e, respectively). To estimate the germ line in-del mutation rate, the genomic region surrounding the TALEN targeted site was amplified from genome DNAs of randomly selected 24 G1 lines. Original size of the PCR products should be 173 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formations on TALEN targeted site. Asterisks (*) show the putative in-del introduced lines. M indicates the marker DNA.

    Journal: Scientific Reports

    Article Title: TALEN-mediated homologous recombination in Daphnia magna

    doi: 10.1038/srep18312

    Figure Lengend Snippet: Establishment and use of Dma-lig4 deficient mutant by CRISPR/Cas. ( a ) Schematic gene structure of Dma-lig4 . Dma-lig4 gene is annotated to consist of 8 exons (open boxes). Putative catalytic domain-encoding regions are highlighted in yellow (adenylation domain) and light green (oligonucleotide binding fold domain). gRNA-targeted sites are shown by triangles. Primers used for RT-PCR are shown by black arrows. Scale bar indicates 0.2 kb. ( b ) PAGE analyses around gRNA #2 target site of putative Dma-lig4 mutants. The genomic region surrounding gRNA #2 target site was amplified from genome DNAs of randomly selected 18 G1 lines. The original size of the PCR products should be 145 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formation on the gRNA #2 targeted site. Asterisks (*) show the putative monoallelic and/or biallelic mutants. M indicates the marker DNA. ( c ) Genotype of Dma-lig4 biallelic mutant strain. Deduced amino acid sequences of wild type or Dma-lig4 mutant #14 are shown under the genome DNA sequences. gRNA #2 targeted sequences and Protospacer Adjacent Motif (PAM) are lined. Especially, gRNA #2 targeted sequences are highlighted in bold. Lower case indicates the introduced in-del mutations into mutant #14 genome. As shown in red, a premature termination codon was introduced into both Dma-lig4 alleles in the mutant #14 genome. ( d , e ) PAGE analyses of G1 offspring from TALEN and targeting plasmid injection using ey Δ877/Δ1 strain with or without Dma-ey deficiency (d, e, respectively). To estimate the germ line in-del mutation rate, the genomic region surrounding the TALEN targeted site was amplified from genome DNAs of randomly selected 24 G1 lines. Original size of the PCR products should be 173 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formations on TALEN targeted site. Asterisks (*) show the putative in-del introduced lines. M indicates the marker DNA.

    Article Snippet: Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA).

    Techniques: Mutagenesis, CRISPR, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Migration, Marker, Plasmid Preparation, Injection