in fusion hd cloning kit  (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:
    None
    Size:
    100 Rxns
    Category:
    In Fusion Cloning for high throughput workflows In Fusion Cloning Cloning
    Buy from Supplier


    Structured Review

    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 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/product/TaKaRa
    Average 99 stars, based on 2075 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning kit - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants"

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

    Journal: Plant Methods

    doi: 10.1186/s13007-017-0236-9

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter"

    Article Title: Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-020-01385-2

    a Construction of the expression vector pSix1 for Thermus strains. The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using Eco RI and Nhe I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein ( sip ) promoter region [ 12 ] were cloned into pYK596 using an In-Fusion cloning kit. The Xho I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1. To complete the Miller assay, a thermostable β-galactosidase gene from the Thermus sp. A4 was cloned downstream of the sip promoter of pSix1 to yield βgal/pSix1. b Sequence of pSix1. The − 35 and − 10 regions of the sip promoter are underlined. The experimentally determined transcription initiation site (+1) is indicated in bold, and the putative ribosome binding site (rbs) is underlined. Restriction sites in the multi-cloning site and 6× histidine tag sequences are indicated above the sequence
    Figure Legend Snippet: a Construction of the expression vector pSix1 for Thermus strains. The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using Eco RI and Nhe I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein ( sip ) promoter region [ 12 ] were cloned into pYK596 using an In-Fusion cloning kit. The Xho I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1. To complete the Miller assay, a thermostable β-galactosidase gene from the Thermus sp. A4 was cloned downstream of the sip promoter of pSix1 to yield βgal/pSix1. b Sequence of pSix1. The − 35 and − 10 regions of the sip promoter are underlined. The experimentally determined transcription initiation site (+1) is indicated in bold, and the putative ribosome binding site (rbs) is underlined. Restriction sites in the multi-cloning site and 6× histidine tag sequences are indicated above the sequence

    Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Derivative Assay, Inverse PCR, Sequencing, Binding Assay

    3) Product Images from "Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis"

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159740

    Characterization of the recombinant PulB of F . tularensis SCHU P9. (A) The plasmid pCold TF contains a lac operator, the cold shock protein A ( cspA ) 5' untranslated region (5' UTR), a translation enhancing element (TEE), a 6x His-tag sequence, the trigger factor sequence, protease cleavage sites, and a multiple cloning site (MCS) downstream of the cspA promoter. This plasmid, which is a cold shock expression vector, can express the target protein fused to a trigger factor under the control of the cold shock protein A ( cspA ) promoter and lac operator. In this study, the pulB gene fused with an N-terminal TEE sequence and a C-terminal 6x his-tag sequence was amplified from F . tularensis SCHU P9 DNA and then inserted into pCold TF plasmid lacking trigger factor, protease sites, and MCS using the In-Fusion HD Cloning Kit. (B) The expression and purification of the recombinant PulB. PulB expression was induced by IPTG in E . coli BL21(DE3) transformed with the pCold TF-pulB plasmid. Recombinant PulB was purified from the bacterial lysates using an AKTA start system equipped with a HisTrap HP column. Lane M, marker proteins (kDa); lane 1, E . coli BL21(DE3) lysates; lane 2, purified recombinant PulB. (C) The products of pullulan hydrolysis catalyzed by recombinant PulB are shown. Pullulan (P), maltotriose (G3), maltose (G2), and glucose (G1) were incubated in pH 6.2 phosphate buffer with (+) or without (–) the recombinant PulB at 37°C for 24 h. After this incubation, the samples were immediately heat denatured. The samples were subjected to TLC analysis using 2-propanol/acetic acid/water (4:1:1, vol/vol/vol) as the solvent system. (D and E) Effects of pH (D) and temperature (E) on recombinant PulB activity are shown. Recombinant PulB was incubated with 0.25% pullulan at 37°C for 12 h and then immediately heat denatured at 94°C for 15 min. The hydrolyzed products were measured in triplicate using the DNS method. After the background was subtracted from the data, the maximal relative activity was determined at pH 6.2 (D) and 37°C (E). Mean ± SD of relative activity are shown. The optimal pH was determined from a curve fitting (Gaussian) by GraphPad Prism software.
    Figure Legend Snippet: Characterization of the recombinant PulB of F . tularensis SCHU P9. (A) The plasmid pCold TF contains a lac operator, the cold shock protein A ( cspA ) 5' untranslated region (5' UTR), a translation enhancing element (TEE), a 6x His-tag sequence, the trigger factor sequence, protease cleavage sites, and a multiple cloning site (MCS) downstream of the cspA promoter. This plasmid, which is a cold shock expression vector, can express the target protein fused to a trigger factor under the control of the cold shock protein A ( cspA ) promoter and lac operator. In this study, the pulB gene fused with an N-terminal TEE sequence and a C-terminal 6x his-tag sequence was amplified from F . tularensis SCHU P9 DNA and then inserted into pCold TF plasmid lacking trigger factor, protease sites, and MCS using the In-Fusion HD Cloning Kit. (B) The expression and purification of the recombinant PulB. PulB expression was induced by IPTG in E . coli BL21(DE3) transformed with the pCold TF-pulB plasmid. Recombinant PulB was purified from the bacterial lysates using an AKTA start system equipped with a HisTrap HP column. Lane M, marker proteins (kDa); lane 1, E . coli BL21(DE3) lysates; lane 2, purified recombinant PulB. (C) The products of pullulan hydrolysis catalyzed by recombinant PulB are shown. Pullulan (P), maltotriose (G3), maltose (G2), and glucose (G1) were incubated in pH 6.2 phosphate buffer with (+) or without (–) the recombinant PulB at 37°C for 24 h. After this incubation, the samples were immediately heat denatured. The samples were subjected to TLC analysis using 2-propanol/acetic acid/water (4:1:1, vol/vol/vol) as the solvent system. (D and E) Effects of pH (D) and temperature (E) on recombinant PulB activity are shown. Recombinant PulB was incubated with 0.25% pullulan at 37°C for 12 h and then immediately heat denatured at 94°C for 15 min. The hydrolyzed products were measured in triplicate using the DNS method. After the background was subtracted from the data, the maximal relative activity was determined at pH 6.2 (D) and 37°C (E). Mean ± SD of relative activity are shown. The optimal pH was determined from a curve fitting (Gaussian) by GraphPad Prism software.

    Techniques Used: Recombinant, Plasmid Preparation, Sequencing, Clone Assay, Expressing, Amplification, Purification, Transformation Assay, Marker, Incubation, Thin Layer Chromatography, Activity Assay, Software

    4) Product Images from "Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis"

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159740

    Characterization of the recombinant PulB of F . tularensis SCHU P9. (A) The plasmid pCold TF contains a lac operator, the cold shock protein A ( cspA ) 5' untranslated region (5' UTR), a translation enhancing element (TEE), a 6x His-tag sequence, the trigger factor sequence, protease cleavage sites, and a multiple cloning site (MCS) downstream of the cspA promoter. This plasmid, which is a cold shock expression vector, can express the target protein fused to a trigger factor under the control of the cold shock protein A ( cspA ) promoter and lac operator. In this study, the pulB gene fused with an N-terminal TEE sequence and a C-terminal 6x his-tag sequence was amplified from F . tularensis SCHU P9 DNA and then inserted into pCold TF plasmid lacking trigger factor, protease sites, and MCS using the In-Fusion HD Cloning Kit. (B) The expression and purification of the recombinant PulB. PulB expression was induced by IPTG in E . coli BL21(DE3) transformed with the pCold TF-pulB plasmid. Recombinant PulB was purified from the bacterial lysates using an AKTA start system equipped with a HisTrap HP column. Lane M, marker proteins (kDa); lane 1, E . coli BL21(DE3) lysates; lane 2, purified recombinant PulB. (C) The products of pullulan hydrolysis catalyzed by recombinant PulB are shown. Pullulan (P), maltotriose (G3), maltose (G2), and glucose (G1) were incubated in pH 6.2 phosphate buffer with (+) or without (–) the recombinant PulB at 37°C for 24 h. After this incubation, the samples were immediately heat denatured. The samples were subjected to TLC analysis using 2-propanol/acetic acid/water (4:1:1, vol/vol/vol) as the solvent system. (D and E) Effects of pH (D) and temperature (E) on recombinant PulB activity are shown. Recombinant PulB was incubated with 0.25% pullulan at 37°C for 12 h and then immediately heat denatured at 94°C for 15 min. The hydrolyzed products were measured in triplicate using the DNS method. After the background was subtracted from the data, the maximal relative activity was determined at pH 6.2 (D) and 37°C (E). Mean ± SD of relative activity are shown. The optimal pH was determined from a curve fitting (Gaussian) by GraphPad Prism software.
    Figure Legend Snippet: Characterization of the recombinant PulB of F . tularensis SCHU P9. (A) The plasmid pCold TF contains a lac operator, the cold shock protein A ( cspA ) 5' untranslated region (5' UTR), a translation enhancing element (TEE), a 6x His-tag sequence, the trigger factor sequence, protease cleavage sites, and a multiple cloning site (MCS) downstream of the cspA promoter. This plasmid, which is a cold shock expression vector, can express the target protein fused to a trigger factor under the control of the cold shock protein A ( cspA ) promoter and lac operator. In this study, the pulB gene fused with an N-terminal TEE sequence and a C-terminal 6x his-tag sequence was amplified from F . tularensis SCHU P9 DNA and then inserted into pCold TF plasmid lacking trigger factor, protease sites, and MCS using the In-Fusion HD Cloning Kit. (B) The expression and purification of the recombinant PulB. PulB expression was induced by IPTG in E . coli BL21(DE3) transformed with the pCold TF-pulB plasmid. Recombinant PulB was purified from the bacterial lysates using an AKTA start system equipped with a HisTrap HP column. Lane M, marker proteins (kDa); lane 1, E . coli BL21(DE3) lysates; lane 2, purified recombinant PulB. (C) The products of pullulan hydrolysis catalyzed by recombinant PulB are shown. Pullulan (P), maltotriose (G3), maltose (G2), and glucose (G1) were incubated in pH 6.2 phosphate buffer with (+) or without (–) the recombinant PulB at 37°C for 24 h. After this incubation, the samples were immediately heat denatured. The samples were subjected to TLC analysis using 2-propanol/acetic acid/water (4:1:1, vol/vol/vol) as the solvent system. (D and E) Effects of pH (D) and temperature (E) on recombinant PulB activity are shown. Recombinant PulB was incubated with 0.25% pullulan at 37°C for 12 h and then immediately heat denatured at 94°C for 15 min. The hydrolyzed products were measured in triplicate using the DNS method. After the background was subtracted from the data, the maximal relative activity was determined at pH 6.2 (D) and 37°C (E). Mean ± SD of relative activity are shown. The optimal pH was determined from a curve fitting (Gaussian) by GraphPad Prism software.

    Techniques Used: Recombinant, Plasmid Preparation, Sequencing, Clone Assay, Expressing, Amplification, Purification, Transformation Assay, Marker, Incubation, Thin Layer Chromatography, Activity Assay, Software

    5) Product Images from "HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors"

    Article Title: HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors

    Journal: PeerJ

    doi: 10.7717/peerj.1060

    Cloning strategy for plasmids encoding Halo-FGFs. DNA encoding HaloTag was inserted 5′ of the FGF2 coding sequence with the In-Fusion HD enzyme. Subsequently, a NotI cleavage site was added 5′ to the BamHI site and other FGFs were exchanged into the plasmid using the digestion-ligation cloning method. A cartoon structure of Halo-FGF is presented in the middle of this figure.
    Figure Legend Snippet: Cloning strategy for plasmids encoding Halo-FGFs. DNA encoding HaloTag was inserted 5′ of the FGF2 coding sequence with the In-Fusion HD enzyme. Subsequently, a NotI cleavage site was added 5′ to the BamHI site and other FGFs were exchanged into the plasmid using the digestion-ligation cloning method. A cartoon structure of Halo-FGF is presented in the middle of this figure.

    Techniques Used: Clone Assay, Sequencing, Plasmid Preparation, Ligation

    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: 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: Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter
    Article Snippet: .. These amplified fragments were cloned into pYK596 at the Nhe I (2852) and Eco RI (992) sites using the In-Fusion HD cloning kit (TaKaRa-bio). .. The resultant plasmid was named pSix0 ( p lasmid for s ilica- i nducible e x pression, 6700 bp).

    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.

    Article Title: Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter
    Article Snippet: .. These amplified fragments were cloned into pYK596 at the Nhe I (2852) and Eco RI (992) sites using the In-Fusion HD cloning kit (TaKaRa-bio). .. The resultant plasmid was named pSix0 ( p lasmid for s ilica- i nducible e x pression, 6700 bp).

    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 2233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    a Construction of the expression vector pSix1 for Thermus strains. The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using Eco RI and Nhe I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein ( sip ) promoter region [ 12 ] were cloned into pYK596 using an In-Fusion cloning kit. The Xho I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1. To complete the Miller assay, a thermostable β-galactosidase gene from the Thermus sp. A4 was cloned downstream of the sip promoter of pSix1 to yield βgal/pSix1. b Sequence of pSix1. The − 35 and − 10 regions of the sip promoter are underlined. The experimentally determined transcription initiation site (+1) is indicated in bold, and the putative ribosome binding site (rbs) is underlined. Restriction sites in the multi-cloning site and 6× histidine tag sequences are indicated above the sequence

    Journal: Microbial Cell Factories

    Article Title: Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

    doi: 10.1186/s12934-020-01385-2

    Figure Lengend Snippet: a Construction of the expression vector pSix1 for Thermus strains. The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using Eco RI and Nhe I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein ( sip ) promoter region [ 12 ] were cloned into pYK596 using an In-Fusion cloning kit. The Xho I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1. To complete the Miller assay, a thermostable β-galactosidase gene from the Thermus sp. A4 was cloned downstream of the sip promoter of pSix1 to yield βgal/pSix1. b Sequence of pSix1. The − 35 and − 10 regions of the sip promoter are underlined. The experimentally determined transcription initiation site (+1) is indicated in bold, and the putative ribosome binding site (rbs) is underlined. Restriction sites in the multi-cloning site and 6× histidine tag sequences are indicated above the sequence

    Article Snippet: These amplified fragments were cloned into pYK596 at the Nhe I (2852) and Eco RI (992) sites using the In-Fusion HD cloning kit (TaKaRa-bio).

    Techniques: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Derivative Assay, Inverse PCR, Sequencing, Binding Assay

    Implementation of TALE–VAS binary expression systems in vivo. a Schematic representation of the TALE–VAS constructs used in flies. The elav 1.8kb , ase 0.8kb , mhc 2.4kb , and repo 1.9kb enhancers were cloned upstream of TALE 1, 3, 4 , and the corresponding 3× VAS sequences were placed in front of Citrine, mCherry, and Cerulean. b – i TALE–VAS-based targeted gene expression in single tissues. Insets show imaging location within each target tissue (red frame). b Dorsal view of elav 1.8kb - TALE 1 > VAS 1 - Citrine-HA third instar larval ventral nerve cord (VNC) stained with anti-HA (transgene expression) and anti-Elav antibody (neuronal marker). c Close up illustrating the overlap between Citrine-HA and nuclear Elav staining in neurons (yellow dashed line and arrow). Some Elav-positive cells lack Citrine-HA expression (yellow asterisk). d Ventral view of ase 0.8kb - TALE 3 > VAS 3 - V5-mCherry third instar larval VNC showing mCherry expression in neuroblasts (red) and Prospero staining of ganglion mother cells (anti-Pros antibody, green). e Detailed, high magnification of a single neuroblast (yellow dashed outline and arrow) and surrounding ganglion mother cells (green). f Third instar larval body wall musculature from mhc 2.4kb - TALE 3 > VAS 3 - V5-mCherry at low magnification. g High-magnification sarcomere morphology showing alternating bands of mCherry expression and F-actin characteristic of striated muscles. h repo 1.9kb - TALE 4 > VAS 4 FLAG-Cerulean third instar larval VNC stained with an anti-GFP antibody (transgene expression) and anti-Repo (glia marker). i Detailed view of subperineural glia showing cells that stain positive for both Cerulean and Repo (yellow dashed outline and arrow) express only Cerulean (blue diamond) or only Repo (yellow asterisk). Scale bars = 50 µm ( b , d , f , h ), 10 µm ( c , e , g , i )

    Journal: Nature Communications

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

    doi: 10.1038/s41467-017-01592-3

    Figure Lengend Snippet: Implementation of TALE–VAS binary expression systems in vivo. a Schematic representation of the TALE–VAS constructs used in flies. The elav 1.8kb , ase 0.8kb , mhc 2.4kb , and repo 1.9kb enhancers were cloned upstream of TALE 1, 3, 4 , and the corresponding 3× VAS sequences were placed in front of Citrine, mCherry, and Cerulean. b – i TALE–VAS-based targeted gene expression in single tissues. Insets show imaging location within each target tissue (red frame). b Dorsal view of elav 1.8kb - TALE 1 > VAS 1 - Citrine-HA third instar larval ventral nerve cord (VNC) stained with anti-HA (transgene expression) and anti-Elav antibody (neuronal marker). c Close up illustrating the overlap between Citrine-HA and nuclear Elav staining in neurons (yellow dashed line and arrow). Some Elav-positive cells lack Citrine-HA expression (yellow asterisk). d Ventral view of ase 0.8kb - TALE 3 > VAS 3 - V5-mCherry third instar larval VNC showing mCherry expression in neuroblasts (red) and Prospero staining of ganglion mother cells (anti-Pros antibody, green). e Detailed, high magnification of a single neuroblast (yellow dashed outline and arrow) and surrounding ganglion mother cells (green). f Third instar larval body wall musculature from mhc 2.4kb - TALE 3 > VAS 3 - V5-mCherry at low magnification. g High-magnification sarcomere morphology showing alternating bands of mCherry expression and F-actin characteristic of striated muscles. h repo 1.9kb - TALE 4 > VAS 4 FLAG-Cerulean third instar larval VNC stained with an anti-GFP antibody (transgene expression) and anti-Repo (glia marker). i Detailed view of subperineural glia showing cells that stain positive for both Cerulean and Repo (yellow dashed outline and arrow) express only Cerulean (blue diamond) or only Repo (yellow asterisk). Scale bars = 50 µm ( b , d , f , h ), 10 µm ( c , e , g , i )

    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).

    Techniques: Expressing, In Vivo, Construct, Clone Assay, Imaging, Staining, Marker

    High-specificity multiplex transgene expression in Drosophila nervous system. a Diagrammatic representation of the experimental framework used for TALE–VAS-mediated parallel transgene expression in multiple tissues in vivo. Tissue-specific expression of three independent transgenes is achieved in the F0 generation by crossing combinatorial driver and responder lines. b Simultaneous expression of mCherry in neuroblasts (red), Citrine in neurons (green), and Cerulean in surface glia cells (white) by three independent TALE–VAS pairs in a single Drosophila larval CNS ( elav 1.8kb -TALE 1 , ase 0.8kb -TALE 3 , repo 1.9kb -TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean ; dorsal view of unfixed CNS). c High-magnification imaging of the right optic lobe from the same genotype in b illustrating the three distinct populations of labelled neighbouring cells (neuroblasts, neurons, and glial cells). d High-magnification imaging of a larval segmental nerve showing an axonal bundle (Citrine, green) ensheathed by a glial cell (Cerulean, white). The ase 0.8kb -TALE 3 driver is inactive in this tissue. e The NMJ innervating muscle 6 and 7 from elav 1.8kb -TALE 1 , mhc 2.4kb -TALE 3 , repo 1.9kb - TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean larvae. The post-synaptic muscle field is labelled by mCherry expression (red) and the neuronal pre-synaptic terminal by Citrine (green). The repo 1.9kb - TALE 4 driver is inactive in this tissue. Scale bars = 100 µm ( b ), 25 µm ( c , e ), 10 µm ( d )

    Journal: Nature Communications

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

    doi: 10.1038/s41467-017-01592-3

    Figure Lengend Snippet: High-specificity multiplex transgene expression in Drosophila nervous system. a Diagrammatic representation of the experimental framework used for TALE–VAS-mediated parallel transgene expression in multiple tissues in vivo. Tissue-specific expression of three independent transgenes is achieved in the F0 generation by crossing combinatorial driver and responder lines. b Simultaneous expression of mCherry in neuroblasts (red), Citrine in neurons (green), and Cerulean in surface glia cells (white) by three independent TALE–VAS pairs in a single Drosophila larval CNS ( elav 1.8kb -TALE 1 , ase 0.8kb -TALE 3 , repo 1.9kb -TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean ; dorsal view of unfixed CNS). c High-magnification imaging of the right optic lobe from the same genotype in b illustrating the three distinct populations of labelled neighbouring cells (neuroblasts, neurons, and glial cells). d High-magnification imaging of a larval segmental nerve showing an axonal bundle (Citrine, green) ensheathed by a glial cell (Cerulean, white). The ase 0.8kb -TALE 3 driver is inactive in this tissue. e The NMJ innervating muscle 6 and 7 from elav 1.8kb -TALE 1 , mhc 2.4kb -TALE 3 , repo 1.9kb - TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean larvae. The post-synaptic muscle field is labelled by mCherry expression (red) and the neuronal pre-synaptic terminal by Citrine (green). The repo 1.9kb - TALE 4 driver is inactive in this tissue. Scale bars = 100 µm ( b ), 25 µm ( c , e ), 10 µm ( d )

    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).

    Techniques: Multiplex Assay, Expressing, In Vivo, Imaging

    Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Journal: Nucleic Acids Research

    Article Title: In-Fusion BioBrick assembly and re-engineering

    doi: 10.1093/nar/gkq179

    Figure Lengend Snippet: Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Article Snippet: The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit.

    Techniques: Ligation, Plasmid Preparation, Purification, Sequencing, Polymerase Chain Reaction, Amplification, Gel Extraction