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
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    Structured Review

    TaKaRa in fusion hd cloning kit clontech takara
    In Fusion Cloning offers a one step high efficiency and high throughput cloning method for projects that require a large number of cloning reactions such as expression screening studies and library construction
    https://www.bioz.com/result/in fusion hd cloning kit clontech takara/product/TaKaRa
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning kit clontech takara - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: .. Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter. .. Rat IL-10 was PCR cloned from rat splenocyte cDNA, as previously described.

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio). ..

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB). ..

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: .. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S. .. The two fragments were ligated using the In-Fusion HD Cloning Kit.

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally epitope-tagged full-length Drosophila Kat80 were created by PCR amplification of the CDS of full-length Kat80 (amino acids 1–819) from the BDGP cDNA clone LD44201 (Drosophila Genomics Resource Center) with the CDS of Myc or FLAG engineered onto the 5’ end of the PCR product.

    Article Title: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
    Article Snippet: .. The pMAL-TEV-MCS41 vector was created as follows: pET-41-a (Novagen) multiple cloning site between SacI to XhoI was amplified by PCR and cloned into SacI –HindIII sites into the pMAL-TEV vector by the In-Fusion® HD Cloning kit (TAKARA). .. The pET41-GFP vector was created as follows: GFP CDS from the pSM-GFP (GenBank: U70495.1) was amplified and cloned into the pET-41-a (Novagen) NotI site by the In-Fusion® HD Cloning kit.

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). ..

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Article Title: Cloning, localization and focus formation at DNA damage sites of canine Ku70
    Article Snippet: .. Each cycle consisted of a denaturation step at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 2 min. PCR products were subcloned into the Eco RI and Apa I sites of pEYFP-C1 (pEYFP-canine Ku70 ) using the In-Fusion HD cloning kit (Takara Bio Inc.), and the inserts were validated by sequencing. .. Other PCR primers used in this study were as follows: dX6 Nseq F1: 5′-GCACAAGGGTGAGCAATAAACAATATG-3′, dX6 Nseq R1: 5′-TGGATACACTGGATGCTCATGTCAAAAG-3′, dX6 Cseq F2: 5′-GGTGGGTTGAAGAAGCAGGAG-3′ and dX6 Cseq R2: 5-AAAGCACAGCAAAGTGGGAGG-3′.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions. .. For constructing the PLK1 shRNA-expression plasmid vector, the DNA fragment containing a PLK1 shRNA coding region was cloned into BamHI/SpeI sites of pTRE3G1.

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: .. The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2. .. The URA3 marker in pLUMCL2 was then replaced with the NAT1 marker to generate the NAT1-Clox cassette in the plasmid pLNMCL.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: .. Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase.

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid
    Article Snippet: .. Construction of pDHB operon: The three genes, Ec-lysC V115A:E119S:E250K:E343V , Bs-asd E218Q and Ms-ssr H39R N43H were cloned into the medium-copy pACT3 vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.) and following the user manual instructions. ..

    Amplification:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: For the targeting vector, 5′ and 3′ homology arms (0.71 kb and 0.82 kb, respectively) of PAX2 were amplified from the genomic DNA of iPS cells with the following primers: 5′-TGAATTCTTAGAGAGACACACACCGGG-3′ and 5′-TGCTAGCGGGGAGGCAGAGGAGCGGGA-3′ for the 5′ homology arm, and 5′-AACCTAGATCGGATCTGCACCGTGAGTACCGGCGC-3′ and 5′-ATTACGCCAAGCTTGCTGGCTCTCTCCCTGACTTC-3′ for the 3′ homology arm. .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB). ..

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: A GFP sequence that includes the promoter Ptet was amplified from the pBRgalKGR plasmid (a pBR322 derivative) [ , ] using the pBR_gfp_kanR_Fw and pBR_gfp_kanR_Rv primers. .. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S.

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally epitope-tagged full-length Drosophila Kat80 were created by PCR amplification of the CDS of full-length Kat80 (amino acids 1–819) from the BDGP cDNA clone LD44201 (Drosophila Genomics Resource Center) with the CDS of Myc or FLAG engineered onto the 5’ end of the PCR product.

    Article Title: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
    Article Snippet: .. The pMAL-TEV-MCS41 vector was created as follows: pET-41-a (Novagen) multiple cloning site between SacI to XhoI was amplified by PCR and cloned into SacI –HindIII sites into the pMAL-TEV vector by the In-Fusion® HD Cloning kit (TAKARA). .. The pET41-GFP vector was created as follows: GFP CDS from the pSM-GFP (GenBank: U70495.1) was amplified and cloned into the pET-41-a (Novagen) NotI site by the In-Fusion® HD Cloning kit.

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: A 1681 bp fragment of the human ABCC4 gene was amplified from the genomic DNA of −1508GG and −1508AA homozygous subjects by PCR using the following primers: (forward) 5′- TCTATCGATAGGTAC GGCCATGCTTAGACATAGGCTTA-3′ and (reverse) 5′- GATCGCAGATCTCGA AGAACACGCGTGAGCAGAGGTT-3′. .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA).

    Article Title: Cloning, localization and focus formation at DNA damage sites of canine Ku70
    Article Snippet: This was followed by 40 cycles of PCR amplification. .. Each cycle consisted of a denaturation step at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 2 min. PCR products were subcloned into the Eco RI and Apa I sites of pEYFP-C1 (pEYFP-canine Ku70 ) using the In-Fusion HD cloning kit (Takara Bio Inc.), and the inserts were validated by sequencing.

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2. .. NAT1 was amplified from pJK863 using the primers Clox-NAT1-F and Clox-NAT1-R , and then cloned between the Bpu10I and NheI sites of pLUMCL2 by In-Fusion cloning to create pLNMCL.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase.

    Mass Spectrometry:

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid
    Article Snippet: .. Construction of pDHB operon: The three genes, Ec-lysC V115A:E119S:E250K:E343V , Bs-asd E218Q and Ms-ssr H39R N43H were cloned into the medium-copy pACT3 vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.) and following the user manual instructions. ..

    Synthesized:

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions. .. For constructing the PLK1 shRNA-expression plasmid vector, the DNA fragment containing a PLK1 shRNA coding region was cloned into BamHI/SpeI sites of pTRE3G1.

    Construct:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: Plasmid construction Multi-expression plasmids were constructed in the pMT/V5-His A expression plasmid backbone (Invitrogen) and additional expression cassettes were cloned into unique restriction enzyme sites engineered into regions outside of the copper-inducible expression cassette. .. The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: Plasmids The constitutive, human codon-optimized, Streptococcus pyogenes Cas9 retroviral expression construct (MSCV-hCas9-PGK-Puro, Addgene: #65655) and lentiviral sgRNA expression vector (LRG, Addgene: #65656) were adapted from previous work [ ]. .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909).

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: The ApaI/XbaI region of rrnB operon was subcloned between HindIII and EcoRI sites of pUC18 in order to facilitate the generation of Spinach ribosome constructs (pUC18-AX). .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB).

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. The multi-expression plasmids constructed in this study are shown in .

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: Paragraph title: 2.5. Activity of the ABCC4 Promoter Constructs ... PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA).

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The pTRE3G1 plasmid vector contains the following constructs: reverse tetracycline activator coding region derived from pCMV-TET3G (Takara Bio Inc., Shiga, Japan); an improved variant of the copepod Pontellina plumata green fluorescent protein (max GFP) coding region derived from pmaxGFP (Lonza, Basel, Switzerland); the GAPDH promoter from nucleotides –376 to + 183 relative to the transcription start site; a tetracycline-response element promoter derived from pTRE3G (Takara Bio); 5′ and 3′ miR-155 flunking region derived from pcDNA 6.2-GW/EmGFP-miR (Thermo Fisher Scientific); and a Luciferase shRNA coding region derived from pSingle-tTS-Anti-Luc (Takara Bio Inc.) for control shRNA. .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: Clox Construction A synthetic, codon-optimized cre open reading frame, interrupted by a C. albicans TUB2 intron at codon 135, was designed in silico (Results), constructed by DNA2.0 (Menlo Park, CA, USA) and cloned between the Nhe I and Nco I sites in pLUL2 to generate pLUCL2. .. The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. A plasmid expressing AM1_1557g2 (pET28a_AM1_1557g2) was then constructed with the TaKaRa in-fusion system reagents. pET28a_AM1_1557g2_C304A was generated using the primers 5′-AGAGACGCACATTTAGAGATTTTGGAA-3′ (forward primer) and 5′-TAAATGTGCGTCTCTATAAGATTCTTG-3′ (reverse primer), pET28a_AM1_1557g2, and PrimeSTAR Max Basal Mutagenesis kit reagents (TaKaRa). pET28a_AM1_1557g2_L337N was obtained in the same way as described above with a primer set (5′- CTATCAGAATAACGTCCCACGTCAATG-3′, 5′- ACGTTATTCTGATAGGCTGCCAGCAA-3′).

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid
    Article Snippet: Construction plasmids for biosynthesis of DHB All plasmids constructed and used in this study are listed in . .. Construction of pDHB operon: The three genes, Ec-lysC V115A:E119S:E250K:E343V , Bs-asd E218Q and Ms-ssr H39R N43H were cloned into the medium-copy pACT3 vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.) and following the user manual instructions.

    Real-time Polymerase Chain Reaction:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter. .. AAV vector titer was determined by real-time PCR using a 7500 Fast Real-Time Instrument (Applied Biosystems) with rat IL-10 gene primers: forward, 5′-TAAGCTCCAAGACAAAGGGTG-3′; and reverse, 5′-GTCCTCCAGTCCAGTAGATG-3′.

    Luciferase:

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The pTRE3G1 plasmid vector contains the following constructs: reverse tetracycline activator coding region derived from pCMV-TET3G (Takara Bio Inc., Shiga, Japan); an improved variant of the copepod Pontellina plumata green fluorescent protein (max GFP) coding region derived from pmaxGFP (Lonza, Basel, Switzerland); the GAPDH promoter from nucleotides –376 to + 183 relative to the transcription start site; a tetracycline-response element promoter derived from pTRE3G (Takara Bio); 5′ and 3′ miR-155 flunking region derived from pcDNA 6.2-GW/EmGFP-miR (Thermo Fisher Scientific); and a Luciferase shRNA coding region derived from pSingle-tTS-Anti-Luc (Takara Bio Inc.) for control shRNA. .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Activity Assay:

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: The PAX2 -TALEN plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific) to evaluate their activity. .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: Paragraph title: 2.5. Activity of the ABCC4 Promoter Constructs ... PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA).

    In Silico:

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: Clox Construction A synthetic, codon-optimized cre open reading frame, interrupted by a C. albicans TUB2 intron at codon 135, was designed in silico (Results), constructed by DNA2.0 (Menlo Park, CA, USA) and cloned between the Nhe I and Nco I sites in pLUL2 to generate pLUCL2. .. The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2.

    Expressing:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: All copper-inducible transgenes were created by cloning PCR-amplified CDS into the multiple cloning site of the copper-inducible expression cassette. .. The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The wild-type human DOT1L cDNA was cloned into a lentiviral expression vector with EFS prompter and P2A-linked Puromycin resistance gene. .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909).

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. A plasmid expressing AM1_1557g2 (pET28a_AM1_1557g2) was then constructed with the TaKaRa in-fusion system reagents. pET28a_AM1_1557g2_C304A was generated using the primers 5′-AGAGACGCACATTTAGAGATTTTGGAA-3′ (forward primer) and 5′-TAAATGTGCGTCTCTATAAGATTCTTG-3′ (reverse primer), pET28a_AM1_1557g2, and PrimeSTAR Max Basal Mutagenesis kit reagents (TaKaRa). pET28a_AM1_1557g2_L337N was obtained in the same way as described above with a primer set (5′- CTATCAGAATAACGTCCCACGTCAATG-3′, 5′- ACGTTATTCTGATAGGCTGCCAGCAA-3′).

    Modification:

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: Upon sequence verification, the 5′ homology arm was cloned in the EcoRI-NheI site of the modified HR120PA-1 vector (Systems Biosciences), such that the start codon of PAX2 was replaced with that of TurboGFP , followed by red fluorescent protein (RFP ) and puromycin-resistance gene (Puro ) cassettes flanked by loxP and insulator sequences. .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: Activity of the ABCC4 Promoter Constructs Human mast cells (HMC-1) were cultured in Iscove's Modified Dulbecco's Medium (Gibco, Grand Island, NY, USA). .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA).

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Derivative Assay:

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: Thus, all three plasmids that were produced had a common site of initiation for replication derived from pUC19, and carried a single gene for ampicillin resistance. .. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The pTRE3G1 plasmid vector contains the following constructs: reverse tetracycline activator coding region derived from pCMV-TET3G (Takara Bio Inc., Shiga, Japan); an improved variant of the copepod Pontellina plumata green fluorescent protein (max GFP) coding region derived from pmaxGFP (Lonza, Basel, Switzerland); the GAPDH promoter from nucleotides –376 to + 183 relative to the transcription start site; a tetracycline-response element promoter derived from pTRE3G (Takara Bio); 5′ and 3′ miR-155 flunking region derived from pcDNA 6.2-GW/EmGFP-miR (Thermo Fisher Scientific); and a Luciferase shRNA coding region derived from pSingle-tTS-Anti-Luc (Takara Bio Inc.) for control shRNA. .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Transfection:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter. .. Recombinant dsAAV vectors (dsAAV1-CAG-IL-10) were generated from cultured serum-free medium using polyethylenimine-based triple transfections of HEK293 cells.

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: The PAX2 -TALEN plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific) to evaluate their activity. .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). .. The constructs were transfected into A549, HMC-1, and U937 cells using Lipofectamine (Invitrogen) according to the manufacturer's protocol.

    Ligation:

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB). ..

    Cell Culture:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter. .. Recombinant dsAAV vectors (dsAAV1-CAG-IL-10) were generated from cultured serum-free medium using polyethylenimine-based triple transfections of HEK293 cells.

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: A549, human alveolar type II epithelial-like, and U937, human leukemic monocyte lymphoma cell lines, were cultured in Roswell Park Memorial Institute-1640 Medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin G sodium, and 100 μ g/mL streptomycin sulfate (Gibco) at 37°C in a 5% CO2 incubator. .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA).

    Generated:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Plasmid and Vector Construction The recombinant double-strand AAV vector plasmid pdsAAV-CBA-EGFP was a kind gift from Arun Srivastava (University of Florida College of Medicine). pdsAAV-CAG-EGFP was generated by replacing the chicken beta-actin (CBA) promoter region of pdsAAV-CBA-EGFP with the cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG) promoter region, as previously described. .. Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter.

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: Plasmid construction and production of PLK1 -specific shRNA and siRNA The doxycycline-regulated and RNA polymerase II-inducible shRNA-expression plasmid vector, designated as pTRE3G1, was generated for this study. .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. A plasmid expressing AM1_1557g2 (pET28a_AM1_1557g2) was then constructed with the TaKaRa in-fusion system reagents. pET28a_AM1_1557g2_C304A was generated using the primers 5′-AGAGACGCACATTTAGAGATTTTGGAA-3′ (forward primer) and 5′-TAAATGTGCGTCTCTATAAGATTCTTG-3′ (reverse primer), pET28a_AM1_1557g2, and PrimeSTAR Max Basal Mutagenesis kit reagents (TaKaRa). pET28a_AM1_1557g2_L337N was obtained in the same way as described above with a primer set (5′- CTATCAGAATAACGTCCCACGTCAATG-3′, 5′- ACGTTATTCTGATAGGCTGCCAGCAA-3′).

    DNA Sequencing:

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The sequences of the genes encoding AM1_1557g2, C304A and L337N were verified by DNA sequencing.

    Sequencing:

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: Upon sequence verification, the 5′ homology arm was cloned in the EcoRI-NheI site of the modified HR120PA-1 vector (Systems Biosciences), such that the start codon of PAX2 was replaced with that of TurboGFP , followed by red fluorescent protein (RFP ) and puromycin-resistance gene (Puro ) cassettes flanked by loxP and insulator sequences. .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB). ..

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: A GFP sequence that includes the promoter Ptet was amplified from the pBRgalKGR plasmid (a pBR322 derivative) [ , ] using the pBR_gfp_kanR_Fw and pBR_gfp_kanR_Rv primers. .. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S.

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. To generate GEN1nuc , a 3xNLS sequence (DPKKKRKVDPKKKRKVDPKKKRKV) and a 3xFLAG tag sequence (AGDYKDHDGDYKDHIDYKDDDDK) were added to the C terminus of GEN1 by PCR.

    Article Title: Cloning, localization and focus formation at DNA damage sites of canine Ku70
    Article Snippet: .. Each cycle consisted of a denaturation step at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 2 min. PCR products were subcloned into the Eco RI and Apa I sites of pEYFP-C1 (pEYFP-canine Ku70 ) using the In-Fusion HD cloning kit (Takara Bio Inc.), and the inserts were validated by sequencing. .. Other PCR primers used in this study were as follows: dX6 Nseq F1: 5′-GCACAAGGGTGAGCAATAAACAATATG-3′, dX6 Nseq R1: 5′-TGGATACACTGGATGCTCATGTCAAAAG-3′, dX6 Cseq F2: 5′-GGTGGGTTGAAGAAGCAGGAG-3′ and dX6 Cseq R2: 5-AAAGCACAGCAAAGTGGGAGG-3′.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions. .. The PLK1 shRNA sequence was designed using siDirect version 2.0 software ( http://sidirect2.rnai.jp/ ).

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: .. Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase.

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid
    Article Snippet: Construction of pDHB operon: The three genes, Ec-lysC V115A:E119S:E250K:E343V , Bs-asd E218Q and Ms-ssr H39R N43H were cloned into the medium-copy pACT3 vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.) and following the user manual instructions. .. The forward primer inserted a unique restriction site and RBS in front of the gene of interest, and the reverse primer had a 15 bp overlapping sequence with the next forward primer. lysC-IF-forward and ssr-IF-reverse primers had 15 bp of sequence complementarity with the 5′ and 3′ ends of the plasmid, respectively.

    Affinity Purification:

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Recombinant:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Plasmid and Vector Construction The recombinant double-strand AAV vector plasmid pdsAAV-CBA-EGFP was a kind gift from Arun Srivastava (University of Florida College of Medicine). pdsAAV-CAG-EGFP was generated by replacing the chicken beta-actin (CBA) promoter region of pdsAAV-CBA-EGFP with the cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG) promoter region, as previously described. .. Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter.

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Crocin Bleaching Assay:

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Plasmid and Vector Construction The recombinant double-strand AAV vector plasmid pdsAAV-CBA-EGFP was a kind gift from Arun Srivastava (University of Florida College of Medicine). pdsAAV-CAG-EGFP was generated by replacing the chicken beta-actin (CBA) promoter region of pdsAAV-CBA-EGFP with the cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG) promoter region, as previously described. .. Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter.

    Mutagenesis:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. All point mutations were introduced by a PCR-based site-directed mutagenesis method and all plasmids were fully sequenced.

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: Both the DOT1L VVEL293MM DOT1L and EZH2 TR683KK mutations were introduced by standard PCR mutagenesis. .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909).

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. A plasmid expressing AM1_1557g2 (pET28a_AM1_1557g2) was then constructed with the TaKaRa in-fusion system reagents. pET28a_AM1_1557g2_C304A was generated using the primers 5′-AGAGACGCACATTTAGAGATTTTGGAA-3′ (forward primer) and 5′-TAAATGTGCGTCTCTATAAGATTCTTG-3′ (reverse primer), pET28a_AM1_1557g2, and PrimeSTAR Max Basal Mutagenesis kit reagents (TaKaRa). pET28a_AM1_1557g2_L337N was obtained in the same way as described above with a primer set (5′- CTATCAGAATAACGTCCCACGTCAATG-3′, 5′- ACGTTATTCTGATAGGCTGCCAGCAA-3′).

    Electrophoretic Mobility Shift Assay:

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: When the resulting fragment was denatured and annealed, a clear band shift was observed, indicating the formation of a mismatched duplex resulting from deletions or insertions . .. The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Purification:

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). ..

    Polymerase Chain Reaction:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter. .. Rat IL-10 was PCR cloned from rat splenocyte cDNA, as previously described.

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: .. Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB). ..

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: .. Transgenes encoding NH2 -terminally GFP-tagged deletion mutants of Kat60 were created by PCR amplification of the CDS of GFP and Kat60-ΔMIT or Kat60-AAA and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). .. Transgenes encoding NH2 -terminally epitope-tagged full-length Drosophila Kat80 were created by PCR amplification of the CDS of full-length Kat80 (amino acids 1–819) from the BDGP cDNA clone LD44201 (Drosophila Genomics Resource Center) with the CDS of Myc or FLAG engineered onto the 5’ end of the PCR product.

    Article Title: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
    Article Snippet: .. The pMAL-TEV-MCS41 vector was created as follows: pET-41-a (Novagen) multiple cloning site between SacI to XhoI was amplified by PCR and cloned into SacI –HindIII sites into the pMAL-TEV vector by the In-Fusion® HD Cloning kit (TAKARA). .. The pET41-GFP vector was created as follows: GFP CDS from the pSM-GFP (GenBank: U70495.1) was amplified and cloned into the pET-41-a (Novagen) NotI site by the In-Fusion® HD Cloning kit.

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). ..

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Article Title: Cloning, localization and focus formation at DNA damage sites of canine Ku70
    Article Snippet: .. Each cycle consisted of a denaturation step at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 2 min. PCR products were subcloned into the Eco RI and Apa I sites of pEYFP-C1 (pEYFP-canine Ku70 ) using the In-Fusion HD cloning kit (Takara Bio Inc.), and the inserts were validated by sequencing. .. Other PCR primers used in this study were as follows: dX6 Nseq F1: 5′-GCACAAGGGTGAGCAATAAACAATATG-3′, dX6 Nseq R1: 5′-TGGATACACTGGATGCTCATGTCAAAAG-3′, dX6 Cseq F2: 5′-GGTGGGTTGAAGAAGCAGGAG-3′ and dX6 Cseq R2: 5-AAAGCACAGCAAAGTGGGAGG-3′.

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: .. The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2. .. The URA3 marker in pLUMCL2 was then replaced with the NAT1 marker to generate the NAT1-Clox cassette in the plasmid pLNMCL.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase.

    Positron Emission Tomography:

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909). .. For the recombinant DOT1L KMT domain, wild-type and VVEL293MM mutant cDNA were cloned into the bacterial expression vector pET-22b (Novagen) via sequence- and ligation-Independent cloning, which contained an N-terminal His6 -tag and TEV protease site that allowed for affinity purification and subsequent cleavage of the affinity tag.

    Article Title: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
    Article Snippet: .. The pMAL-TEV-MCS41 vector was created as follows: pET-41-a (Novagen) multiple cloning site between SacI to XhoI was amplified by PCR and cloned into SacI –HindIII sites into the pMAL-TEV vector by the In-Fusion® HD Cloning kit (TAKARA). .. The pET41-GFP vector was created as follows: GFP CDS from the pSM-GFP (GenBank: U70495.1) was amplified and cloned into the pET-41-a (Novagen) NotI site by the In-Fusion® HD Cloning kit.

    cDNA Library Assay:

    Article Title: Cloning, localization and focus formation at DNA damage sites of canine Ku70
    Article Snippet: Cloning of canine Ku70 Oligonucleotide primers used to amplify canine Ku70 cDNA from a male beagle dog cDNA library (Biochain, Newark, CA, U.S.A.) were designed based on the predicted Ku70 genomic sequence of a female boxer dog, Canis lupus familiaris (XM_531714.3). .. Each cycle consisted of a denaturation step at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 2 min. PCR products were subcloned into the Eco RI and Apa I sites of pEYFP-C1 (pEYFP-canine Ku70 ) using the In-Fusion HD cloning kit (Takara Bio Inc.), and the inserts were validated by sequencing.

    Plasmid Preparation:

    Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity
    Article Snippet: Paragraph title: Plasmid construction ... The transgene encoding internally GFP-tagged full-length Kat60 was created by PCR amplification of the CDS of Kat60-MIT (amino acids 1–96), GFP, and Kat60-ΔMIT and fusion of the resulting PCR products using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.).

    Article Title: Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke
    Article Snippet: Paragraph title: Plasmid and Vector Construction ... Using the In-Fusion HD Cloning Kit (TakaRa Bio), the EGFP region of pdsAAV-CAG-EGFP was replaced with rat IL-10 to generate pdsAAV-CAG-IL-10: rat IL-10 driven by the CAG promoter.

    Article Title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
    Article Snippet: The wild-type EHZ2 cDNA was cloned into an MSCV-based vector containing a puromycin-resistance gene and a GFP reporter as previously reported [ ]. .. The PCR cloning procedures were performed using the In-Fusion® HD Cloning Kit (Clontech: #638909).

    Article Title: PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells
    Article Snippet: Paragraph title: Generation of PAX2-TALEN plasmids and the targeting vector ... The 3′ homology arm was cloned in the BamHI-SphI site using an In-fusion HD Cloning Kit (Takara Bio).

    Article Title: Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs
    Article Snippet: Paragraph title: Plasmid construction ... Each Spinach sequence was amplified by PCR and cloned into pUC18-AXP using either an In-Fusion HD Cloning Kit (Clontech) or a Quick Ligation Kit (NEB).

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: Paragraph title: Plasmid construction ... The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S.

    Article Title: Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
    Article Snippet: .. The pMAL-TEV-MCS41 vector was created as follows: pET-41-a (Novagen) multiple cloning site between SacI to XhoI was amplified by PCR and cloned into SacI –HindIII sites into the pMAL-TEV vector by the In-Fusion® HD Cloning kit (TAKARA). .. The pET41-GFP vector was created as follows: GFP CDS from the pSM-GFP (GenBank: U70495.1) was amplified and cloned into the pET-41-a (Novagen) NotI site by the In-Fusion® HD Cloning kit.

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). ..

    Article Title: Spatial control of the GEN1 Holliday junction resolvase ensures genome stability
    Article Snippet: .. Plasmids The GEN1 truncation constructs (full-length GEN1, GEN11–220 , GEN1221–444 , GEN1444–527 , GEN1526–662 , GEN1526–683 and GEN1662–908 ) were generated by PCR and cloned in-frame into a modified pDONR221 vector (Life Technologies) encoding a C-terminal GFP tag using an In-Fusion HD cloning kit (Clontech). .. The coding sequences were then shuttled by Gateway recombination (Life Technologies) into a modified pcDNA5/FRT vector (Life Technologies) with a Gateway cassette RfB cloned into the EcoRV site.

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: Paragraph title: Plasmid construction and production of PLK1 -specific shRNA and siRNA ... The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: .. The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2. .. The URA3 marker in pLUMCL2 was then replaced with the NAT1 marker to generate the NAT1-Clox cassette in the plasmid pLNMCL.

    Article Title: A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina
    Article Snippet: .. Plasmid Construction The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). .. The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase.

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid
    Article Snippet: .. Construction of pDHB operon: The three genes, Ec-lysC V115A:E119S:E250K:E343V , Bs-asd E218Q and Ms-ssr H39R N43H were cloned into the medium-copy pACT3 vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.) and following the user manual instructions. ..

    Software:

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions. .. The PLK1 shRNA sequence was designed using siDirect version 2.0 software ( http://sidirect2.rnai.jp/ ).

    shRNA:

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: Paragraph title: Plasmid construction and production of PLK1 -specific shRNA and siRNA ... The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

    Agarose Gel Electrophoresis:

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: .. PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). ..

    Spectrophotometry:

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). .. Plasmid DNAs were prepared from these constructs using the Endo-Free Plasmid Maxi Kit (Qiagen, Hilden, Germany), and the concentration and purity were assessed by UV spectrophotometry and agarose gel electrophoresis.

    Produced:

    Article Title: A reduced genome decreases the host carrying capacity for foreign DNA
    Article Snippet: Thus, all three plasmids that were produced had a common site of initiation for replication derived from pUC19, and carried a single gene for ampicillin resistance. .. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2) , resulting in a 2,334-bp-long fragment, the same length as pUC-S.

    Concentration Assay:

    Article Title: A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics
    Article Snippet: PCR products were gel purified using an Agarose Gel Purification Kit (GeneAll Biotechnology, Seoul, Korea) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA) after digestion with KpnІ and XhoІ (Takara, Shuzo, Japan) using the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). .. Plasmid DNAs were prepared from these constructs using the Endo-Free Plasmid Maxi Kit (Qiagen, Hilden, Germany), and the concentration and purity were assessed by UV spectrophotometry and agarose gel electrophoresis.

    Marker:

    Article Title: New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
    Article Snippet: The CaMET3 promoter region (1336 bp) was then PCR-amplified using Infusion cloning primers Clox-MET3p-F and Clox-MET3p-R ( ) and cloned between the Nhe I and Xma I sites in pLUCL2 in front of the cre gene using an In-Fusion HD cloning kit according to the manufacturer’s instructions (Clontech, California, USA) to generate the URA3-Clox cassette in the plasmid pLUMCL2. .. The URA3 marker in pLUMCL2 was then replaced with the NAT1 marker to generate the NAT1-Clox cassette in the plasmid pLNMCL.

    Variant Assay:

    Article Title: Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma
    Article Snippet: The pTRE3G1 plasmid vector contains the following constructs: reverse tetracycline activator coding region derived from pCMV-TET3G (Takara Bio Inc., Shiga, Japan); an improved variant of the copepod Pontellina plumata green fluorescent protein (max GFP) coding region derived from pmaxGFP (Lonza, Basel, Switzerland); the GAPDH promoter from nucleotides –376 to + 183 relative to the transcription start site; a tetracycline-response element promoter derived from pTRE3G (Takara Bio); 5′ and 3′ miR-155 flunking region derived from pcDNA 6.2-GW/EmGFP-miR (Thermo Fisher Scientific); and a Luciferase shRNA coding region derived from pSingle-tTS-Anti-Luc (Takara Bio Inc.) for control shRNA. .. The DNA fragments were synthesized using the GeneArt Strings system (Thermo Fisher Scientific) and cloned into SalI-PciI restriction enzyme sites of pcDNA3.1 (Thermo Fisher Scientific) using the In-Fusion HD cloning kit (Takara Bio Inc.), according to the manufacturer’s instructions.

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    TaKaRa in fusion hd cloning kit
    Overview of <t>In-Fusion</t> enzyme-based <t>cloning</t> of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® <t>HD</t> cloning <t>kit</t> with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
    In Fusion Hd Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1623 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa transcription factor overexpression
    Western blot to determine effect of formaldehyde and degree of <t>overexpression</t> compared to wild type
    Transcription Factor Overexpression, supplied by TaKaRa, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

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

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

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

    Journal: Plant Methods

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

    doi: 10.1186/s13007-017-0236-9

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

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

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

    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

    Western blot to determine effect of formaldehyde and degree of overexpression compared to wild type

    Journal: Methods (San Diego, Calif.)

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method

    doi: 10.1016/j.ymeth.2014.10.029

    Figure Lengend Snippet: Western blot to determine effect of formaldehyde and degree of overexpression compared to wild type

    Article Snippet: Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech)

    Techniques: Western Blot, Over Expression