in fusion hd cloning kit clontech takara  (TaKaRa)

 
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    Name:
    In Fusion HD Cloning Kit
    Description:
    In Fusion Cloning offers a one step high efficiency and high throughput cloning method for projects that require a large number of cloning reactions such as expression screening studies and library construction
    Catalog Number:
    639650
    Price:
    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 clontech takara
    In Fusion Cloning offers a one step high efficiency and high throughput cloning method for projects that require a large number of cloning reactions such as expression screening studies and library construction
    https://www.bioz.com/result/in fusion hd cloning kit clontech takara/product/TaKaRa
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning kit clontech takara - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

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

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio). .. The PCR product including PB transposon IRs (p3EIR) was created using p3E1.2 as a template, primer5, and primer6.

    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: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech). .. To generate a mutated version of Alhid lacking two potential MAPK phosphorylation sites ( Alhid Ala 2 ), the clone M100 containing the complete Alhid ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using AH666-AH667, and a 650 bp fragment using AH668-AH669.

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

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: .. Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit ( , ). .. The proprietary In-Fusion enzyme with exonuclease activity fuses together any PCR product with a linearized vector into a circular plasmid when the fragments share at least 15 bp of homology on both ends.

    Article Title: TALEN-mediated homologous recombination in Daphnia magna
    Article Snippet: .. Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA). .. Microinjection In vitro synthesised RNAs and/or donor DNAs were injected into eyΔ877/Δ1 mutant eggs according to established procedures .

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Bacmid preparation, generation of virus stocks and infection of Sf9 cells were performed according to the manufacturer's instructions.

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: .. The In-Fusion PCR cloning kit was then used to insert cDNA of each transcription factor coding region into the Pst I-linearized vector. .. To verify the DNA sequence of the prepared vectors, we used the BigDye Terminator v3.1 Cycle Sequencing Kit with a 3130 Genetic Analyzer (Life Technologies, Grand Island, NY).

    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.

    Luciferase:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio). .. The expression cassette including the firefly luciferase gene under CMV promoter control and neomycin-resistance gene under SV40 promoter control was amplified by PCR using pCMV-luc as a template, primer7, and primer8.

    Reporter Assay:

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: Paragraph title: In Vitro Minigene Splicing Reporter Assay ... 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).

    Construct:

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: To construct the p35S-HH3-MRGFP-RFP plasmid, the p35S-HH1-MRGFP-RFP plasmid was used as a scaffold sequence and template. .. 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).

    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. .. This vector was used as a template for making gRNA constructs.

    Article Title: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech). .. To generate a mutated version of Alhid lacking two potential MAPK phosphorylation sites ( Alhid Ala 2 ), the clone M100 containing the complete Alhid ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using AH666-AH667, and a 650 bp fragment using AH668-AH669.

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: For F8 exon 13 minigene constructs, a 1,182-bp fragment including 202 bp of intron 12, the exon 13, and 770 bp of intron 13 was amplified from the two propositus and control DNA using specific primers ( ) and PrimeSTAR GXL DNA polymerase (Clonetech). .. 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).

    Incubation:

    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 plates were incubated overnight at 37°C, and colonies resistant to ampicillin were selected.

    Amplification:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: The pNVU1 plasmid was also amplified using the pCold-F for pulB and pCold-R for pulB primer pair ( ). .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: To create pFerH-PBTP, PB transposase ORF was amplified using p3E1.2 (a gift from Prof. Hajime Mori, Kyoto Institute of Technology, Kyoto, Japan) as a template, and primer3 and primer4. .. The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio).

    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: Similarly, the plasmid pEn-Chimera backbone was amplified using 240 nM of primers 5-PENchimera and 3-PENchimera (each 2 µl of a 1.2 µM stock, Table ), 20 pg plasmid pEN-Chimera (1 µl 20 pg/µl) and 5 µl 2 × CloneAmp™ HiFi PCR Premix using PCR conditions as above. .. 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.

    Article Title: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech). .. To generate a mutated version of Alhid lacking two potential MAPK phosphorylation sites ( Alhid Ala 2 ), the clone M100 containing the complete Alhid ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using AH666-AH667, and a 650 bp fragment using AH668-AH669.

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit ( , ). .. To assemble two BioBricks, one PCR-amplified BioBrick needs to have homology on each end with the second PCR-amplified BioBrick (vector amplified with the BioBrick) to allow for the fragments to be fused together in the In-Fusion reaction ( b).

    Article Title: TALEN-mediated homologous recombination in Daphnia magna
    Article Snippet: To generate the targeting plasmid, 1.5 kb of target-homologous region nearly centred at the TALEN-targeted site was first amplified from genomic DNA by using PrimeSTAR (Takara Bio) and cloned into pCR-BluntII-TOPO vectors (Life Technologies), which resulted in the creation of pCR-BluntII-Dma-ey1.5kb. .. 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).

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. For the purification of Flag-tagged Chd1 proteins, ~2 × 108 Sf9 cells were infected with the amplified virus stock and allowed to express protein for 3 days.

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: For F8 exon 13 minigene constructs, a 1,182-bp fragment including 202 bp of intron 12, the exon 13, and 770 bp of intron 13 was amplified from the two propositus and control DNA using specific primers ( ) and PrimeSTAR GXL DNA polymerase (Clonetech). .. 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).

    Activity Assay:

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit ( , ). .. The proprietary In-Fusion enzyme with exonuclease activity fuses together any PCR product with a linearized vector into a circular plasmid when the fragments share at least 15 bp of homology on both ends.

    Infection:

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Bacmid preparation, generation of virus stocks and infection of Sf9 cells were performed according to the manufacturer's instructions.

    Expressing:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: Complementation of the ΔpulB mutant with wild-type pulB To complement the ΔpulB mutant, the pNVU1 expression plasmid [ ], designated p(cont) in this study, was modified. .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio). .. The expression cassette including the firefly luciferase gene under CMV promoter control and neomycin-resistance gene under SV40 promoter control was amplified by PCR using pCMV-luc as a template, primer7, and primer8.

    Modification:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: Complementation of the ΔpulB mutant with wild-type pulB To complement the ΔpulB mutant, the pNVU1 expression plasmid [ ], designated p(cont) in this study, was modified. .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Transformation Assay:

    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: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech). .. To generate a mutated version of Alhid lacking two potential MAPK phosphorylation sites ( Alhid Ala 2 ), the clone M100 containing the complete Alhid ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using AH666-AH667, and a 650 bp fragment using AH668-AH669.

    Ligation:

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: The PCR product was elongated further by a second round of PCR using primer pair T-DNA RB F/HRz Xma I R2, followed by ligation into the Xma I and Hind III sites of the vector p35S-HH1-MRGFP-RFP to yield an intermediate plasmid. .. 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).

    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.

    Cell Culture:

    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. .. A single colony was cultured in 50 ml of LB broth, and then the plasmid purified using NucleoBond PC 100 (Macherey-Nagel GmbH & Co.) was verified by determining its sequence.

    Generated:

    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). .. The p35St-MRGFP-RFP , which contained a truncated 35S promoter, was generated by removing the sequence between the transcriptional initiation site of promoter and the hammerhead ribozyme of p35S-HH1-MRGFP-RFP .

    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).

    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). .. In order to discontinue the T-tract of the AluY element, five substitutions (T > C) were generated in pTB2ex13WT through site-directed mutagenesis using the QuickChange XL reagent (Stratagene) according to the manufacturer’s instructions.

    DNA Sequencing:

    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: 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.

    Polymerase Chain Reaction:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: The PCR products were purified using NucleoSpin Gel and PCR Clean-up (Machery-Nagel). .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio). .. The PCR product including PB transposon IRs (p3EIR) was created using p3E1.2 as a template, primer5, and primer6.

    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 PCR products were purified using NucleoSpin Gel and PCR Clean-up columns (Machery-Nagel, Duren, Germany). .. 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.

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
    Article Snippet: Similarly, the plasmid pEn-Chimera backbone was amplified using 240 nM of primers 5-PENchimera and 3-PENchimera (each 2 µl of a 1.2 µM stock, Table ), 20 pg plasmid pEN-Chimera (1 µl 20 pg/µl) and 5 µl 2 × CloneAmp™ HiFi PCR Premix using PCR conditions as above. .. 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.

    Article Title: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech). .. To generate a mutated version of Alhid lacking two potential MAPK phosphorylation sites ( Alhid Ala 2 ), the clone M100 containing the complete Alhid ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using AH666-AH667, and a 650 bp fragment using AH668-AH669.

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

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: .. Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit ( , ). .. The proprietary In-Fusion enzyme with exonuclease activity fuses together any PCR product with a linearized vector into a circular plasmid when the fragments share at least 15 bp of homology on both ends.

    Article Title: TALEN-mediated homologous recombination in Daphnia magna
    Article Snippet: .. Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA). .. Microinjection In vitro synthesised RNAs and/or donor DNAs were injected into eyΔ877/Δ1 mutant eggs according to established procedures .

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Bacmid preparation, generation of virus stocks and infection of Sf9 cells were performed according to the manufacturer's instructions.

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: .. The In-Fusion PCR cloning kit was then used to insert cDNA of each transcription factor coding region into the Pst I-linearized vector. .. To verify the DNA sequence of the prepared vectors, we used the BigDye Terminator v3.1 Cycle Sequencing Kit with a 3130 Genetic Analyzer (Life Technologies, Grand Island, NY).

    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.

    Recombinant:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: Paragraph title: Production and purification of recombinant PulB ... 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.

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: Paragraph title: Synthesis and purification of recombinant proteins ... The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector .

    Mutagenesis:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: Paragraph title: Complementation of the ΔpulB mutant with wild-type pulB ... The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    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: In-Fusion BioBrick assembly and re-engineering
    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. .. We describe the success rate and mutation rate of In-Fusion assembled genetic circuits using various homology and primer lengths.

    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.

    Purification:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: The PCR products were purified using NucleoSpin Gel and PCR Clean-up (Machery-Nagel). .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: The PCR fragment was purified, and self-ligated using Mighty Cloning Kit (blunt end) (Takara Bio, Ōtsu, Japan). .. The PCR product was cloned into pFerH-mcs using In-Fusion PCR cloning kit (Takara Bio).

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: Paragraph title: Production and purification of recombinant PulB ... 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.

    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: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: Paragraph title: Synthesis and purification of recombinant proteins ... The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector .

    Sequencing:

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: To construct the p35S-HH3-MRGFP-RFP plasmid, the p35S-HH1-MRGFP-RFP plasmid was used as a scaffold sequence and template. .. 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).

    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. .. A single colony was cultured in 50 ml of LB broth, and then the plasmid purified using NucleoBond PC 100 (Macherey-Nagel GmbH & Co.) was verified by determining its sequence.

    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 sequence of the pTB2ex13WT_5mut was 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.

    Plasmid Preparation:

    Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
    Article Snippet: The pNVU1 plasmid was also amplified using the pCold-F for pulB and pCold-R for pulB primer pair ( ). .. The two amplicons, which contained a 15-bp overlap with each other, were connected using an In-Fusion HD Cloning Kit (Clontech Laboratories).

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
    Article Snippet: To construct the p35S-HH3-MRGFP-RFP plasmid, the p35S-HH1-MRGFP-RFP plasmid was used as a scaffold sequence and template. .. 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).

    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: Paragraph title: Construction of plasmid pEn-Chimera-ccdB ... 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.

    Article Title: Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa
    Article Snippet: We composed our constructs in the cloning shuttle vector pSLfa1180fa ( ). .. From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry Fse I and Asc I sites ( fa -sites). pSL af_Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into Xba I cut pSL af_tTA_af (#1215) , using the In-Fusion PCR cloning Kit (Clontech).

    Article Title: In-Fusion BioBrick assembly and re-engineering
    Article Snippet: The latter two methods involve overlapping oligonucleotides with a PCR-amplified vector ( ) or extending overlapping PCR products ( ) and do not use subsequent enzymatic treatment. .. Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit ( , ).

    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).

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Bacmid preparation, generation of virus stocks and infection of Sf9 cells were performed according to the manufacturer's instructions.

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: .. The In-Fusion PCR cloning kit was then used to insert cDNA of each transcription factor coding region into the Pst I-linearized vector. .. To verify the DNA sequence of the prepared vectors, we used the BigDye Terminator v3.1 Cycle Sequencing Kit with a 3130 Genetic Analyzer (Life Technologies, Grand Island, NY).

    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.

    In Vitro:

    Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
    Article Snippet: Paragraph title: In Vitro Minigene Splicing Reporter Assay ... 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).

    Produced:

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: Synthesis and purification of recombinant proteins Recombinant Chd1 proteins were produced using the baculovirus system (Bac-to-Bac®, Life Technologies). .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector .

    FLAG-tag:

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Bacmid preparation, generation of virus stocks and infection of Sf9 cells were performed according to the manufacturer's instructions.

    BAC Assay:

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: Synthesis and purification of recombinant proteins Recombinant Chd1 proteins were produced using the baculovirus system (Bac-to-Bac®, Life Technologies). .. The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector .

    Lysis:

    Article Title: Embryonic stem cell differentiation requires full length Chd1
    Article Snippet: The coding sequences of full-length mouse Chd1 (aa 1-1711), and two truncated versions of Chd1 (aa 101-1711 and aa 150-1711) were cloned using the in-Fusion PCR Cloning Kit (Clontech) into the Sal I site of a Flag-tag encoding pFastBac1 vector . .. Cells were collected, washed with PBS and resuspended in 10 ml Lysis Buffer (20 mM Tris/HCl pH 7.9, 10 mM NaCl, 4 mM MgCl2 , 0.4 mM EDTA, 20% glycerol, 0.4 mM PMSF, 2 mM DTT).

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    TaKaRa infusion pcr cloning kit
    MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the <t>pGL3</t> -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to <t>PCR.</t> E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.
    Infusion Pcr Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: Revelation of p53-independent Function of MTA1 in DNA Damage Response via Modulation of the p21WAF1-Proliferating Cell Nuclear Antigen Pathway *

    doi: 10.1074/jbc.M109.079095

    Figure Lengend Snippet: MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Article Snippet: Mutant mouse p21WAF1 promoter lacking the MTA1 binding region was constructed by cloning the PCR product amplified from the mouse p21WAF1 promoter (region from −1860 to −632) using the forward primer (5′-TAGCCCGGGCTCGAGAGATATCCGTTCGTTCAAACTAAGACTCC-3′) and reverse primer (5′-CCGGAATGCCAAGCTTGAGGCACGAGGGGCGTTACAGGTTCAA-3′), and then cloned into a pGL3 basic vector after digesting with XhoI and HindIII using the Clontech Infusion PCR cloning kit (Clontech).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Purification, Polymerase Chain Reaction

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

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

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

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

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

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

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

    Journal: Virology Journal

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

    doi: 10.1186/s12985-017-0776-7

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

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

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

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

    Journal: Virology Journal

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

    doi: 10.1186/s12985-017-0776-7

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

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

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

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

    Journal: American Journal of Human Genetics

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

    doi: 10.1016/j.ajhg.2017.12.010

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

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

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