infusion hd cloning kit  (TaKaRa)


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    Name:
    In Fusion HD Cloning Kit
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    Catalog Number:
    638933
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    500 Rxns
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    Structured Review

    TaKaRa infusion hd cloning kit
    The steps of constructing of pACYC177‐JEVpr4DENV2 plasmid. (A) The structure of pACYC177 plasmid. (B) The pACYC177 plasmid was digested by Sac II and Cla I and purified; specific PCR products were purified. (C) Construction of a part of pACYC177‐DENV2(1–5505) cDNA infectious clone using an In‐Fusion™ <t>HD</t> <t>cloning</t> <t>kit.</t> (D) The pACYC177‐DENV2(1–5505) plasmid was digested by Xho I and Cla I and purified; specific PCR products were purified. (E) Construction of a full‐length pACYC177‐DENV2(10723) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (F) pACYC177‐DENV2(10723) plasmid was digested by Sac II and Spe I. The construction of pACYC177‐JEVpr4DENV2 plasmid is shown

    https://www.bioz.com/result/infusion hd cloning kit/product/TaKaRa
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    infusion hd cloning kit - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research"

    Article Title: Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research

    Journal: The Journal of Gene Medicine

    doi: 10.1002/jgm.3297

    The steps of constructing of pACYC177‐JEVpr4DENV2 plasmid. (A) The structure of pACYC177 plasmid. (B) The pACYC177 plasmid was digested by Sac II and Cla I and purified; specific PCR products were purified. (C) Construction of a part of pACYC177‐DENV2(1–5505) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (D) The pACYC177‐DENV2(1–5505) plasmid was digested by Xho I and Cla I and purified; specific PCR products were purified. (E) Construction of a full‐length pACYC177‐DENV2(10723) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (F) pACYC177‐DENV2(10723) plasmid was digested by Sac II and Spe I. The construction of pACYC177‐JEVpr4DENV2 plasmid is shown
    Figure Legend Snippet: The steps of constructing of pACYC177‐JEVpr4DENV2 plasmid. (A) The structure of pACYC177 plasmid. (B) The pACYC177 plasmid was digested by Sac II and Cla I and purified; specific PCR products were purified. (C) Construction of a part of pACYC177‐DENV2(1–5505) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (D) The pACYC177‐DENV2(1–5505) plasmid was digested by Xho I and Cla I and purified; specific PCR products were purified. (E) Construction of a full‐length pACYC177‐DENV2(10723) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (F) pACYC177‐DENV2(10723) plasmid was digested by Sac II and Spe I. The construction of pACYC177‐JEVpr4DENV2 plasmid is shown

    Techniques Used: Plasmid Preparation, Purification, Polymerase Chain Reaction, Clone Assay

    Related Articles

    Clone Assay:

    Article Title: TALEN-mediated homologous recombination in Daphnia magna
    Article Snippet: Second, the 60 bp core attP sequences with Bam HI as the insert were generated by PCR from pTA-attP (Addgene plasmid 18939 ) with KOD plus (TOYOBO, Osaka, Japan). .. 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). .. Microinjection In vitro synthesised RNAs and/or donor DNAs were injected into eyΔ877/Δ1 mutant eggs according to established procedures .

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. .. 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. ..

    Article Title: A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus
    Article Snippet: The amino acid sequences of Arabidopsis and Populus CaM/CML proteins were used to build a phylogenetic tree using Maximum Likelihood method of the MEGA 7.0.25 software. .. The coding sequences of the 21 putative interactors and 26 calmodulin/calmodulin-like CaM genes were cloned into pGADT7 (Clontech) vector and PdIQD10 into pGBKT7 (Clontech) vector using In-Fusion® Advantage PCR Cloning Kit (Clontech). .. The generated constructs were transformed into yeast Y2H Gold (Clontech) competent cells using FastTM Yeast Transformation (G-Biosciences Cat. #GZ-1) kit.

    Article Title: HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors
    Article Snippet: .. Enzymes for cloning were from: NcoI, BamHI and T4 ligase (NEB, Hitchin, UK); KOD Hot Start DNA polymerase (Merck, Hertfordshire, UK); In-Fusion® HD Cloning Kit (Clontech, Takara Bio Europe SAS, Saint-Germain-en-Laye, France). .. Bacterial cells: DH5α , BL21 (DE3) pLysS and SoluBL21 were a gift from Olga Mayans, University of Liverpool.

    Article Title: Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system
    Article Snippet: The pBFv-AeU6 backbone plasmid was modified from the pBFv-U6.2 plasmid obtained from the Cas9 reagents provided by the Fly Stocks of the National Institute of Genetics (NIG-Fly) in Japan ( ). .. The Drosophila U6.2 promoter was replaced with an Ae. aegypti U6 promoter to generate a pBFv-AeU6 backbone plasmid using an In-Fusion HD Cloning Kit (Clontech). a. .. We took 520 base pairs (bp) of the AeU6 promoter from the AAEL017763 locus of Ae. aegypti CRITICAL: Primers designed for In-Fusion HD cloning must have two characteristics: (1) the 5′ end of the primer must contain 15 homologous bases to coincide with the end of the DNA fragment to which it will be joined (i.e., the vector or another insert); (2) the 3′ end of the primer must contain sequences specific to the target gene. i. ii. iii.The first PCR products act as a template for secondary PCR using AeU6-gRNA-F1 and AeU6-gRNA-R2 primers. b. Linearize the pBFv-U6.2 plasmid via double digestion with Eco RI/Not I (NEB). i. ii.

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]
    Article Snippet: Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC , white perch Lr8− , and the translated striped bass ovary transcriptome ( , ). .. A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. .. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen).

    Article Title: Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research
    Article Snippet: .. The ligation reaction was performed with a Clontech Infusion HD cloning kit. .. The restriction sites Xho I and ClaI were used to double digest pACYC177‐DENV2 (1–5505) plasmid and create ends that could be connected to two fragments of DENV 5426–10,723 (DENV2S3, DENV2S4).

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis
    Article Snippet: The pCB301-2X35S-Nos plasmid was linearized by Stu I and Sma I double digestion, which removes the sequence between the transcription initiation site of 35S promoter and the multiple cloning sites immediately before the HDV ribozyme sequence. .. The linearized plasmid was recovered and assembled precisely with the SYNV cDNA by using an In-Fusion HD PCR Cloning Kit (Clontech, Japan). .. The resulting plasmid (pSYNV) was sequenced to confirm its authenticity and correct assembly, and was expected to transcribe the full-length SYNV antigenome with the exact 5′ end by 35S promoter and the exact 3′ end processed by the HDV ribozyme cleavages.

    Polymerase Chain Reaction:

    Article Title: TALEN-mediated homologous recombination in Daphnia magna
    Article Snippet: Second, the 60 bp core attP sequences with Bam HI as the insert were generated by PCR from pTA-attP (Addgene plasmid 18939 ) with KOD plus (TOYOBO, Osaka, Japan). .. 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). .. Microinjection In vitro synthesised RNAs and/or donor DNAs were injected into eyΔ877/Δ1 mutant eggs according to established procedures .

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. .. 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. ..

    Article Title: A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus
    Article Snippet: The amino acid sequences of Arabidopsis and Populus CaM/CML proteins were used to build a phylogenetic tree using Maximum Likelihood method of the MEGA 7.0.25 software. .. The coding sequences of the 21 putative interactors and 26 calmodulin/calmodulin-like CaM genes were cloned into pGADT7 (Clontech) vector and PdIQD10 into pGBKT7 (Clontech) vector using In-Fusion® Advantage PCR Cloning Kit (Clontech). .. The generated constructs were transformed into yeast Y2H Gold (Clontech) competent cells using FastTM Yeast Transformation (G-Biosciences Cat. #GZ-1) kit.

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]
    Article Snippet: Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC , white perch Lr8− , and the translated striped bass ovary transcriptome ( , ). .. A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. .. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen).

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis
    Article Snippet: The pCB301-2X35S-Nos plasmid was linearized by Stu I and Sma I double digestion, which removes the sequence between the transcription initiation site of 35S promoter and the multiple cloning sites immediately before the HDV ribozyme sequence. .. The linearized plasmid was recovered and assembled precisely with the SYNV cDNA by using an In-Fusion HD PCR Cloning Kit (Clontech, Japan). .. The resulting plasmid (pSYNV) was sequenced to confirm its authenticity and correct assembly, and was expected to transcribe the full-length SYNV antigenome with the exact 5′ end by 35S promoter and the exact 3′ end processed by the HDV ribozyme cleavages.

    Chick Chorioallantoic Membrane Assay:

    Article Title: A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus
    Article Snippet: The amino acid sequences of Arabidopsis and Populus CaM/CML proteins were used to build a phylogenetic tree using Maximum Likelihood method of the MEGA 7.0.25 software. .. The coding sequences of the 21 putative interactors and 26 calmodulin/calmodulin-like CaM genes were cloned into pGADT7 (Clontech) vector and PdIQD10 into pGBKT7 (Clontech) vector using In-Fusion® Advantage PCR Cloning Kit (Clontech). .. The generated constructs were transformed into yeast Y2H Gold (Clontech) competent cells using FastTM Yeast Transformation (G-Biosciences Cat. #GZ-1) kit.

    Plasmid Preparation:

    Article Title: A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus
    Article Snippet: The amino acid sequences of Arabidopsis and Populus CaM/CML proteins were used to build a phylogenetic tree using Maximum Likelihood method of the MEGA 7.0.25 software. .. The coding sequences of the 21 putative interactors and 26 calmodulin/calmodulin-like CaM genes were cloned into pGADT7 (Clontech) vector and PdIQD10 into pGBKT7 (Clontech) vector using In-Fusion® Advantage PCR Cloning Kit (Clontech). .. The generated constructs were transformed into yeast Y2H Gold (Clontech) competent cells using FastTM Yeast Transformation (G-Biosciences Cat. #GZ-1) kit.

    Article Title: Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system
    Article Snippet: The pBFv-AeU6 backbone plasmid was modified from the pBFv-U6.2 plasmid obtained from the Cas9 reagents provided by the Fly Stocks of the National Institute of Genetics (NIG-Fly) in Japan ( ). .. The Drosophila U6.2 promoter was replaced with an Ae. aegypti U6 promoter to generate a pBFv-AeU6 backbone plasmid using an In-Fusion HD Cloning Kit (Clontech). a. .. We took 520 base pairs (bp) of the AeU6 promoter from the AAEL017763 locus of Ae. aegypti CRITICAL: Primers designed for In-Fusion HD cloning must have two characteristics: (1) the 5′ end of the primer must contain 15 homologous bases to coincide with the end of the DNA fragment to which it will be joined (i.e., the vector or another insert); (2) the 3′ end of the primer must contain sequences specific to the target gene. i. ii. iii.The first PCR products act as a template for secondary PCR using AeU6-gRNA-F1 and AeU6-gRNA-R2 primers. b. Linearize the pBFv-U6.2 plasmid via double digestion with Eco RI/Not I (NEB). i. ii.

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]
    Article Snippet: Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC , white perch Lr8− , and the translated striped bass ovary transcriptome ( , ). .. A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. .. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen).

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis
    Article Snippet: The pCB301-2X35S-Nos plasmid was linearized by Stu I and Sma I double digestion, which removes the sequence between the transcription initiation site of 35S promoter and the multiple cloning sites immediately before the HDV ribozyme sequence. .. The linearized plasmid was recovered and assembled precisely with the SYNV cDNA by using an In-Fusion HD PCR Cloning Kit (Clontech, Japan). .. The resulting plasmid (pSYNV) was sequenced to confirm its authenticity and correct assembly, and was expected to transcribe the full-length SYNV antigenome with the exact 5′ end by 35S promoter and the exact 3′ end processed by the HDV ribozyme cleavages.

    Expressing:

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]
    Article Snippet: Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC , white perch Lr8− , and the translated striped bass ovary transcriptome ( , ). .. A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. .. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen).

    Ligation:

    Article Title: Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research
    Article Snippet: .. The ligation reaction was performed with a Clontech Infusion HD cloning kit. .. The restriction sites Xho I and ClaI were used to double digest pACYC177‐DENV2 (1–5505) plasmid and create ends that could be connected to two fragments of DENV 5426–10,723 (DENV2S3, DENV2S4).

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    TaKaRa in fusion hd pcr cloning kit
    Expression of GFP engineered into rSYNV. (A) Diagram showing insertion of the GFP gene between the N and P genes of the rSYNV antigenome to produce rSYNV-GFP. The recombinant vector transcribes GFP mRNA under the control of a duplicated N/P gene junction sequence. (B) Time course of GFP foci appearing in N . benthamiana leaves after agroinfiltration with rSYNV-GFP derivatives. N . benthamiana leaves were agroinfiltrated with bacterial mixtures containing plasmids encoding the rSYNV-GFP agRNA, the N, P, L proteins and the VSR suppressor proteins, and photographed by fluorescence microscopy at 6, 9, 12 and 15 dpi. The white arrow indicates GFP entering a leaf vein. Scale bar, 200 μm. (C) Symptoms of rSYNV- and rSYNV-GFP-infected plants. Photographs were taken at 30 dpi under white light (upper panels) and ultraviolet (UV) light (bottom panels). (D) Immunoblot analysis of <t>SYNV</t> proteins and GFP expression in plants systemically infected with rSYNV-GFP. Total proteins extracted from rSYNV (lane 1) and rSYNV-GFP (lane 2) infected plants were analyzed by Western blotting with antibodies against disrupted SYNV virions (α:SYNV) or GFP (α:GFP). (E) Stable maintenance of GFP during serial passages of rSYNV-GFP in N . benthamiana . rSYNV-GFP passaged to healthy plants by mechanical inoculation, and after 5 serial passages, the infected plants were photographed at 16 dpi under white (upper panels) and UV light (bottom panels). (F) Total RNAs and proteins were extracted from upper infected leaves of each plant passage and analyzed by <t>RT-PCR</t> using GFP-specific primers or by immmunoblotting (WB) with αSYNV and αGFP antibodies. Numbers 1 to 5 on the top of the panel represent passages No. 1 to 5, respectively. Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control.
    In Fusion Hd Pcr Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in fusion hd pcr cloning kit/product/TaKaRa
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    in fusion hd pcr cloning kit - by Bioz Stars, 2021-07
    99/100 stars
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    Expression of GFP engineered into rSYNV. (A) Diagram showing insertion of the GFP gene between the N and P genes of the rSYNV antigenome to produce rSYNV-GFP. The recombinant vector transcribes GFP mRNA under the control of a duplicated N/P gene junction sequence. (B) Time course of GFP foci appearing in N . benthamiana leaves after agroinfiltration with rSYNV-GFP derivatives. N . benthamiana leaves were agroinfiltrated with bacterial mixtures containing plasmids encoding the rSYNV-GFP agRNA, the N, P, L proteins and the VSR suppressor proteins, and photographed by fluorescence microscopy at 6, 9, 12 and 15 dpi. The white arrow indicates GFP entering a leaf vein. Scale bar, 200 μm. (C) Symptoms of rSYNV- and rSYNV-GFP-infected plants. Photographs were taken at 30 dpi under white light (upper panels) and ultraviolet (UV) light (bottom panels). (D) Immunoblot analysis of SYNV proteins and GFP expression in plants systemically infected with rSYNV-GFP. Total proteins extracted from rSYNV (lane 1) and rSYNV-GFP (lane 2) infected plants were analyzed by Western blotting with antibodies against disrupted SYNV virions (α:SYNV) or GFP (α:GFP). (E) Stable maintenance of GFP during serial passages of rSYNV-GFP in N . benthamiana . rSYNV-GFP passaged to healthy plants by mechanical inoculation, and after 5 serial passages, the infected plants were photographed at 16 dpi under white (upper panels) and UV light (bottom panels). (F) Total RNAs and proteins were extracted from upper infected leaves of each plant passage and analyzed by RT-PCR using GFP-specific primers or by immmunoblotting (WB) with αSYNV and αGFP antibodies. Numbers 1 to 5 on the top of the panel represent passages No. 1 to 5, respectively. Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control.

    Journal: PLoS Pathogens

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

    doi: 10.1371/journal.ppat.1005223

    Figure Lengend Snippet: Expression of GFP engineered into rSYNV. (A) Diagram showing insertion of the GFP gene between the N and P genes of the rSYNV antigenome to produce rSYNV-GFP. The recombinant vector transcribes GFP mRNA under the control of a duplicated N/P gene junction sequence. (B) Time course of GFP foci appearing in N . benthamiana leaves after agroinfiltration with rSYNV-GFP derivatives. N . benthamiana leaves were agroinfiltrated with bacterial mixtures containing plasmids encoding the rSYNV-GFP agRNA, the N, P, L proteins and the VSR suppressor proteins, and photographed by fluorescence microscopy at 6, 9, 12 and 15 dpi. The white arrow indicates GFP entering a leaf vein. Scale bar, 200 μm. (C) Symptoms of rSYNV- and rSYNV-GFP-infected plants. Photographs were taken at 30 dpi under white light (upper panels) and ultraviolet (UV) light (bottom panels). (D) Immunoblot analysis of SYNV proteins and GFP expression in plants systemically infected with rSYNV-GFP. Total proteins extracted from rSYNV (lane 1) and rSYNV-GFP (lane 2) infected plants were analyzed by Western blotting with antibodies against disrupted SYNV virions (α:SYNV) or GFP (α:GFP). (E) Stable maintenance of GFP during serial passages of rSYNV-GFP in N . benthamiana . rSYNV-GFP passaged to healthy plants by mechanical inoculation, and after 5 serial passages, the infected plants were photographed at 16 dpi under white (upper panels) and UV light (bottom panels). (F) Total RNAs and proteins were extracted from upper infected leaves of each plant passage and analyzed by RT-PCR using GFP-specific primers or by immmunoblotting (WB) with αSYNV and αGFP antibodies. Numbers 1 to 5 on the top of the panel represent passages No. 1 to 5, respectively. Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control.

    Article Snippet: The linearized plasmid was recovered and assembled precisely with the SYNV cDNA by using an In-Fusion HD PCR Cloning Kit (Clontech, Japan).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining

    Recovery of recombinant SYNV in N . benthamiana . (A) Schematic representation of the SYNV infectious cDNA clone and supporting plasmids. The pSYNV plasmid is designed for transcription to yield the SYNV antigenome (ag) RNA and contains the full-length SYNV cDNA positioned between a truncated CaMV double 35S promoter (2X35S) and the HDV ribozyme sequence in the pCB301 plasmid. Note that the SYNV gene order is shown in the antigenome sense. The pGD-N, pGD-P and pGD-L supporting plasmids encode the viral N, P and L core NC proteins, respectively, and the pGD-VSRs encode the TBSV p19, BSMV γB, and TEV P1/HC-Pro suppressors of RNA silencing (VSRs). le: leader; tr: trailer; Rz: ribozyme; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator. (B) Symptoms of N . benthamiana plants systemically infected with rSYNV. Agrobacterium cultures containing the pSYNV, pGD-N, pGD-P, pGD-L and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Infected plants showing stunting and typical vein clearing symptoms were photographed at 35 days post infiltration (dpi) along with a mock control (uninfected healthy plant). (C) Detection of viral structural proteins in wtSYNV- and rSYNV-infected N . benthamiana plants. Total protein samples were analyzed by Western blotting with anti-SYNV virion antibody. The Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control. The positions of the SYNV G, N, M and P proteins are indicated along the left side of the gel and the numbers along the right side are protein size markers in KDa. (D) Restriction enzyme site identification of wtSYNV and rSYNV cDNAs. Total RNAs were extracted from wtSYNV- and rSYNV-infected plants and used as templates for reverse transcription PCR (RT-PCR). The wtSYNV and rSYNV RT-PCR products (∼1500 bp) were digested with Bsm BI or Apa I and analyzed on 1.5% agarose gels. Positions of DNA size markers (M) are shown in base pairs.

    Journal: PLoS Pathogens

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

    doi: 10.1371/journal.ppat.1005223

    Figure Lengend Snippet: Recovery of recombinant SYNV in N . benthamiana . (A) Schematic representation of the SYNV infectious cDNA clone and supporting plasmids. The pSYNV plasmid is designed for transcription to yield the SYNV antigenome (ag) RNA and contains the full-length SYNV cDNA positioned between a truncated CaMV double 35S promoter (2X35S) and the HDV ribozyme sequence in the pCB301 plasmid. Note that the SYNV gene order is shown in the antigenome sense. The pGD-N, pGD-P and pGD-L supporting plasmids encode the viral N, P and L core NC proteins, respectively, and the pGD-VSRs encode the TBSV p19, BSMV γB, and TEV P1/HC-Pro suppressors of RNA silencing (VSRs). le: leader; tr: trailer; Rz: ribozyme; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator. (B) Symptoms of N . benthamiana plants systemically infected with rSYNV. Agrobacterium cultures containing the pSYNV, pGD-N, pGD-P, pGD-L and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Infected plants showing stunting and typical vein clearing symptoms were photographed at 35 days post infiltration (dpi) along with a mock control (uninfected healthy plant). (C) Detection of viral structural proteins in wtSYNV- and rSYNV-infected N . benthamiana plants. Total protein samples were analyzed by Western blotting with anti-SYNV virion antibody. The Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control. The positions of the SYNV G, N, M and P proteins are indicated along the left side of the gel and the numbers along the right side are protein size markers in KDa. (D) Restriction enzyme site identification of wtSYNV and rSYNV cDNAs. Total RNAs were extracted from wtSYNV- and rSYNV-infected plants and used as templates for reverse transcription PCR (RT-PCR). The wtSYNV and rSYNV RT-PCR products (∼1500 bp) were digested with Bsm BI or Apa I and analyzed on 1.5% agarose gels. Positions of DNA size markers (M) are shown in base pairs.

    Article Snippet: The linearized plasmid was recovered and assembled precisely with the SYNV cDNA by using an In-Fusion HD PCR Cloning Kit (Clontech, Japan).

    Techniques: Recombinant, Plasmid Preparation, Sequencing, Infection, Western Blot, Staining, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Cloning procedure for the pCR2-TOPO-attP-loxp-Pub-eGFP plasmid using the In-Fusion HD Cloning Kit

    Journal: STAR Protocols

    Article Title: Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system

    doi: 10.1016/j.xpro.2021.100432

    Figure Lengend Snippet: Cloning procedure for the pCR2-TOPO-attP-loxp-Pub-eGFP plasmid using the In-Fusion HD Cloning Kit

    Article Snippet: The Drosophila U6.2 promoter was replaced with an Ae. aegypti U6 promoter to generate a pBFv-AeU6 backbone plasmid using an In-Fusion HD Cloning Kit (Clontech). a.

    Techniques: Clone Assay, Plasmid Preparation

    The steps of constructing of pACYC177‐JEVpr4DENV2 plasmid. (A) The structure of pACYC177 plasmid. (B) The pACYC177 plasmid was digested by Sac II and Cla I and purified; specific PCR products were purified. (C) Construction of a part of pACYC177‐DENV2(1–5505) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (D) The pACYC177‐DENV2(1–5505) plasmid was digested by Xho I and Cla I and purified; specific PCR products were purified. (E) Construction of a full‐length pACYC177‐DENV2(10723) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (F) pACYC177‐DENV2(10723) plasmid was digested by Sac II and Spe I. The construction of pACYC177‐JEVpr4DENV2 plasmid is shown

    Journal: The Journal of Gene Medicine

    Article Title: Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. Antibody‐dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research

    doi: 10.1002/jgm.3297

    Figure Lengend Snippet: The steps of constructing of pACYC177‐JEVpr4DENV2 plasmid. (A) The structure of pACYC177 plasmid. (B) The pACYC177 plasmid was digested by Sac II and Cla I and purified; specific PCR products were purified. (C) Construction of a part of pACYC177‐DENV2(1–5505) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (D) The pACYC177‐DENV2(1–5505) plasmid was digested by Xho I and Cla I and purified; specific PCR products were purified. (E) Construction of a full‐length pACYC177‐DENV2(10723) cDNA infectious clone using an In‐Fusion™ HD cloning kit. (F) pACYC177‐DENV2(10723) plasmid was digested by Sac II and Spe I. The construction of pACYC177‐JEVpr4DENV2 plasmid is shown

    Article Snippet: The ligation reaction was performed with a Clontech Infusion HD cloning kit.

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

    Semi-quantitative RT-PCR of lrp13 and rpl9 in striped bass tissues. The PCRs were terminated after 25, 30, and 35 cycles (indicated at the left).

    Journal: Journal of Lipid Research

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]

    doi: 10.1194/jlr.M050286

    Figure Lengend Snippet: Semi-quantitative RT-PCR of lrp13 and rpl9 in striped bass tissues. The PCRs were terminated after 25, 30, and 35 cycles (indicated at the left).

    Article Snippet: A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R.

    Techniques: Quantitative RT-PCR

    Real-time quantitative RT-PCR of lrp13 and lr8 − (left) and quantitative mass spectrometry of Lrp13 (right) in ESG, MVG, LVG, and PVG striped bass ovary. Vertical bars and data points represent mean values and brackets indicate standard errors of the mean. Mean values bearing the same letter superscript are not significantly different ( P

    Journal: Journal of Lipid Research

    Article Title: Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes [S]

    doi: 10.1194/jlr.M050286

    Figure Lengend Snippet: Real-time quantitative RT-PCR of lrp13 and lr8 − (left) and quantitative mass spectrometry of Lrp13 (right) in ESG, MVG, LVG, and PVG striped bass ovary. Vertical bars and data points represent mean values and brackets indicate standard errors of the mean. Mean values bearing the same letter superscript are not significantly different ( P

    Article Snippet: A portion of cutthroat trout ( Oncorhynchus clarki ) CtLR13+1 (Lrp13) ( ) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R.

    Techniques: Quantitative RT-PCR, Mass Spectrometry