ecori xhoi  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ecori xhoi
    Ecori Xhoi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori xhoi/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ecori xhoi - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: .. Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. For the construction of the mammalian and bacterial expression vectors, the gateway cloning system technology (Thermo Fischer Scientific, USA) was applied.

    Article Title: Altered stoichiometry and nuclear delocalization of NonO and PSF promote cellular senescence
    Article Snippet: Plasmids pCR3.1-HA-NonO was constructed by excising full-length HA-NonO from pBS-HA-NonO1.4 vector with EcoRI/XhoI, and ligating it to EcoRI/XhoI digested pCR3.1 (Invitrogen). pBI-NonO2.4 was generated by excising full length NonO from p5.7NonO with XhoI and ligating it to SalI digested pBI (Clontech). .. GFP-NonO and deletion mutants were created by cloning a NonO cDNA into the pEGFP-C1 vector (Clontech).

    Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis
    Article Snippet: .. Reporter vectors The parental vector pUASt-XBP1-EGFP was a kind gift from H Steller and HD Ryoo (NYU School of Medicine, USA) . pMT-V5-eroGFP was obtained by cloning a EcoRI/XhoI -digested fragment encompassing the coding fragment of roGFP-HDEL from the p28M kar2ss-roGFP2-HDEL vector into the pMT-His-V5 expression vector (Invitrogen). .. The inducible IRE1-EGFP construct pMT-IRE1-Flag-EGFP ( ) was obtained by cloning a PCR product from pcDNA5.1/3xFLAGhIRE1-EGFP with flanking KpnI and NotI sites in the pMT-His-V5 vector.

    Article Title: Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold
    Article Snippet: .. The obtained PCR product was digested with EcoRI-XhoI and cloned into pYD1 vector (Invitrogen) digested with EcoRI-XhoI generating pYD1/FL- iafgp . .. The plasmids pYD1/FL- iafgp or pYD1 (without insert) were transformed into EBY100 competent cells, and cold tolerance assays were performed as mentioned in the Supplemental Methods.

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: The amplified PCR product was digested with XhoI/HindIII and cloned into pPicaGene-Basic vector II (Toyo-ink), which yielded TBX21 -Luc. .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen).

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: The resultant fragment was then cloned into the pGEM-T vector (Promega, Madison, WI, USA) and the sequence was confirmed by sequencing. .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector.

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter. .. The MATCHMAKER two-hybrid system 3 (Clontech) was used according to the supplier’s protocol with the Sox6 bait plasmid, detailed above.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: N-terminal deletion ( i.e. encoded amino acids 5–397, 10–397, or 15–397) and C-terminal deletion mutants of pUL50 ( i.e. encoded amino acids 1–181, 1–150, 1–130, 1–100, or 1–70) were generated by cloning of PCR products. .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP).

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy). .. In this vector cN-II ORF is placed under control of the inducible Gal1 promoter which allows the expression of the protein only when galactose is added to the growth medium.

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: .. DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. For EDIN production, the DNA was cloned using BamHI–EcoRI into pET28a after PCR amplification using oligonucleotides 5′-CCGGATCCGCTGATGTTAAAAATTTCACTGATTTAG-3′ and 5′ GGGAATTCCTATTTTTTAAAAACAATAGCTGTTATT-3′.

    Article Title: BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei
    Article Snippet: .. The BILBO1 ORF and truncations were cloned into the pcDNA3 between HindIII-XbaI sites for BILBO1 full length and EcoRI-XhoI for the truncations or into pcDNA3.1 CT-GFP TOPO (Invitrogen). .. Mutations of the EF-hand domain 1 (D194A (GAT/GcT); N198A (AAC/gcC); D202A (GAC/GcC); D205A (GAC/GcC), the EF-hand domain 2 (D230A (GAC/GcC); N232A (AAC/gcC); E241A (GAA/GcA), and the serine 163 mutations (S163D (TCG/gat) and S163A (TCG/gCG) were done by site-directed mutagenesis following the instructions from the Agilent QuickChange Site-directed Mutagenesis kit.

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: MAPK4, PTEN, and HSP27 cDNAs were cloned by RT–PCR from U2OS cells. .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively.

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: .. The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen). .. The GST-hYSK1 was inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, Buckinghamshire, UK).

    Amplification:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. Open reading frames (ORFs) of human full-length (FL) RIPK4 (NM_020639.2) and kinase-dead RIPK4 (K51R) were amplified from pCR3 VSV-RIPK4 (Meylan et al., 2002) by PCR with primers suitable for gateway cloning.

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. Recombined plasmids were extracted from S. cerevisiae and transformed into E. coli DH5α for amplification.

    Article Title: Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold
    Article Snippet: The full-length iafgp gene was PCR amplified with oligonucleotides 5′-CGGAATTCATGACGACTCTGCTTCGTCTGACT-3′ and 5′-CCGCTCGAGCGCAGCCGCCGTAGCT-3′ containing EcoRI and XhoI sites, respectively, from pGEMT- iafgp plasmid DNA as a template. .. The obtained PCR product was digested with EcoRI-XhoI and cloned into pYD1 vector (Invitrogen) digested with EcoRI-XhoI generating pYD1/FL- iafgp .

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen). .. Tax construct with FLAG-tag added to the N terminus was prepared via PCR amplification of template DNA ( ) with the following primers: Tax forward, 5′-CGCGAATTCATGGCCCACTTCCCAGGGTTT-3′; Tax reverse, 5′-CGCCTCGAGTCAGACTTCTGTTTCACGGAAATGTTTTTC-3′.

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The second recombinant PCR was performed to link the resultant Aβq with a 219 bp of chimeric cDNA that was PCR amplified from pRmHA3-Aβq-leuZ-bio-flag and consisted of a leucine zipper peptide, a flexible linker (RGGASGG), a biotinylation sequence and a flag-tag sequence. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: The derived plasmid was used as the template for amplification by polymerase chain reaction (PCR) using Pfu DNA polymerase. .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector.

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To generate the plasmid pGADT7 (Clontech) for the Co-IP, the amplified PCR fragment encoding the first 108 amino acids of the Prtb gene (GenBank accession no. ), and RT–PCR of the entire gene (168 amino acids), were cloned in-frame into EcoRI–XhoI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: Standard PCR amplification was performed using the template pcDNA-UL50-HA ( ) with oligonucleotide primers purchased from Biomers (Ulm, Germany); sequences of oligonucleotides are given in . .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP).

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: Plasmid and Yeast Strain The cN-II cDNA was amplified by PCR using pET-28c+cNII as template. .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy).

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: .. DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. For EDIN production, the DNA was cloned using BamHI–EcoRI into pET28a after PCR amplification using oligonucleotides 5′-CCGGATCCGCTGATGTTAAAAATTTCACTGATTTAG-3′ and 5′ GGGAATTCCTATTTTTTAAAAACAATAGCTGTTATT-3′.

    Polymerase Chain Reaction:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. Open reading frames (ORFs) of human full-length (FL) RIPK4 (NM_020639.2) and kinase-dead RIPK4 (K51R) were amplified from pCR3 VSV-RIPK4 (Meylan et al., 2002) by PCR with primers suitable for gateway cloning.

    Article Title: Altered stoichiometry and nuclear delocalization of NonO and PSF promote cellular senescence
    Article Snippet: Plasmids pCR3.1-HA-NonO was constructed by excising full-length HA-NonO from pBS-HA-NonO1.4 vector with EcoRI/XhoI, and ligating it to EcoRI/XhoI digested pCR3.1 (Invitrogen). pBI-NonO2.4 was generated by excising full length NonO from p5.7NonO with XhoI and ligating it to SalI digested pBI (Clontech). .. To construct GFP-NonO and all the other mutants, NonO fragments were generated by PCR using a 5′ primer containing a XhoI restriction site and a 3′ primer containing an EcoRI restriction site and template p5.7NonO.

    Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis
    Article Snippet: Reporter vectors The parental vector pUASt-XBP1-EGFP was a kind gift from H Steller and HD Ryoo (NYU School of Medicine, USA) . pMT-V5-eroGFP was obtained by cloning a EcoRI/XhoI -digested fragment encompassing the coding fragment of roGFP-HDEL from the p28M kar2ss-roGFP2-HDEL vector into the pMT-His-V5 expression vector (Invitrogen). .. The inducible IRE1-EGFP construct pMT-IRE1-Flag-EGFP ( ) was obtained by cloning a PCR product from pcDNA5.1/3xFLAGhIRE1-EGFP with flanking KpnI and NotI sites in the pMT-His-V5 vector.

    Article Title: Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold
    Article Snippet: .. The obtained PCR product was digested with EcoRI-XhoI and cloned into pYD1 vector (Invitrogen) digested with EcoRI-XhoI generating pYD1/FL- iafgp . .. The plasmids pYD1/FL- iafgp or pYD1 (without insert) were transformed into EBY100 competent cells, and cold tolerance assays were performed as mentioned in the Supplemental Methods.

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: The amplified PCR product was digested with XhoI/HindIII and cloned into pPicaGene-Basic vector II (Toyo-ink), which yielded TBX21 -Luc. .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen).

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The second recombinant PCR was performed to link the resultant Aβq with a 219 bp of chimeric cDNA that was PCR amplified from pRmHA3-Aβq-leuZ-bio-flag and consisted of a leucine zipper peptide, a flexible linker (RGGASGG), a biotinylation sequence and a flag-tag sequence. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: The derived plasmid was used as the template for amplification by polymerase chain reaction (PCR) using Pfu DNA polymerase. .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector.

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To generate the plasmid pGADT7 (Clontech) for the Co-IP, the amplified PCR fragment encoding the first 108 amino acids of the Prtb gene (GenBank accession no. ), and RT–PCR of the entire gene (168 amino acids), were cloned in-frame into EcoRI–XhoI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. For generating constructs coding for fragments of pUL50 ( i.e. encoded amino acids 1–358, 1–205, 236–358, or 10–169) fused to GFP and β-gal, PCR amplification was performed as described above.

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy). .. In this vector cN-II ORF is placed under control of the inducible Gal1 promoter which allows the expression of the protein only when galactose is added to the growth medium.

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: .. DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. For EDIN production, the DNA was cloned using BamHI–EcoRI into pET28a after PCR amplification using oligonucleotides 5′-CCGGATCCGCTGATGTTAAAAATTTCACTGATTTAG-3′ and 5′ GGGAATTCCTATTTTTTAAAAACAATAGCTGTTATT-3′.

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively. .. HSP27 mutants were generated by the insertion of oligonucleotides (Supplemental Table S2A).

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: .. The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen). .. The GST-hYSK1 was inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, Buckinghamshire, UK).

    Construct:

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. To construct HA-tagged torsinA and torsinAΔE vectors, a single HA tag was introduced at the C terminus of torsinA and torsinAΔE by in vivo recombination in S. cerevisiae following a previously published protocol ( ).

    Article Title: Altered stoichiometry and nuclear delocalization of NonO and PSF promote cellular senescence
    Article Snippet: .. Plasmids pCR3.1-HA-NonO was constructed by excising full-length HA-NonO from pBS-HA-NonO1.4 vector with EcoRI/XhoI, and ligating it to EcoRI/XhoI digested pCR3.1 (Invitrogen). pBI-NonO2.4 was generated by excising full length NonO from p5.7NonO with XhoI and ligating it to SalI digested pBI (Clontech). .. GFP-NonO and deletion mutants were created by cloning a NonO cDNA into the pEGFP-C1 vector (Clontech).

    Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis
    Article Snippet: Reporter vectors The parental vector pUASt-XBP1-EGFP was a kind gift from H Steller and HD Ryoo (NYU School of Medicine, USA) . pMT-V5-eroGFP was obtained by cloning a EcoRI/XhoI -digested fragment encompassing the coding fragment of roGFP-HDEL from the p28M kar2ss-roGFP2-HDEL vector into the pMT-His-V5 expression vector (Invitrogen). .. The inducible IRE1-EGFP construct pMT-IRE1-Flag-EGFP ( ) was obtained by cloning a PCR product from pcDNA5.1/3xFLAGhIRE1-EGFP with flanking KpnI and NotI sites in the pMT-His-V5 vector.

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: Creation of the human Sp1 construct with HA-tag added to the N terminus was accomplished via real-time RT-PCR amplification of human PBMC cDNA with the following primers: Sp1 forward, 5′-CGCGAATTCATGAGCGACCAAGATCACTCCATGGA-3′; Sp1 reverse, 5′-CGCCTCGAGTCAGAAGCCATTGCCACTGATATTAATGGAC-3′. .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen).

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct. .. Both AαQ and AβQ constructs were verified by DNA sequencing.

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To construct the plasmid pGBKT7 (Clontech) as bait for the yeast two-hybrid screen and for the co-immunoprecipitation (Co-IP), an amplified PCR fragment encoding amino acids 139–304 (including the coiled-coil domain) of mouse Sox6 (GenBank accession no. ) was cloned in-frame into EcoRI–SalI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. For generating constructs coding for fragments of pUL50 ( i.e. encoded amino acids 1–358, 1–205, 236–358, or 10–169) fused to GFP and β-gal, PCR amplification was performed as described above.

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: .. DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. For EDIN production, the DNA was cloned using BamHI–EcoRI into pET28a after PCR amplification using oligonucleotides 5′-CCGGATCCGCTGATGTTAAAAATTTCACTGATTTAG-3′ and 5′ GGGAATTCCTATTTTTTAAAAACAATAGCTGTTATT-3′.

    Luciferase:

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively. .. HSP27 mutants were generated by the insertion of oligonucleotides (Supplemental Table S2A).

    Expressing:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: .. Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. For the construction of the mammalian and bacterial expression vectors, the gateway cloning system technology (Thermo Fischer Scientific, USA) was applied.

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: .. The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. To construct HA-tagged torsinA and torsinAΔE vectors, a single HA tag was introduced at the C terminus of torsinA and torsinAΔE by in vivo recombination in S. cerevisiae following a previously published protocol ( ).

    Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis
    Article Snippet: .. Reporter vectors The parental vector pUASt-XBP1-EGFP was a kind gift from H Steller and HD Ryoo (NYU School of Medicine, USA) . pMT-V5-eroGFP was obtained by cloning a EcoRI/XhoI -digested fragment encompassing the coding fragment of roGFP-HDEL from the p28M kar2ss-roGFP2-HDEL vector into the pMT-His-V5 expression vector (Invitrogen). .. The inducible IRE1-EGFP construct pMT-IRE1-Flag-EGFP ( ) was obtained by cloning a PCR product from pcDNA5.1/3xFLAGhIRE1-EGFP with flanking KpnI and NotI sites in the pMT-His-V5 vector.

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector. .. Construction of plasmids expressing truncated NDUFV2 proteins The pcDNA4-NDUFV2 vector was used as the template for generation of its N-terminal deletion constructs (pcDNA4-△1-18 NDUFV2, pcDNA4-△1-32 NDUFV2 and pcDNA4-△1-50 NDUFV2) and C-terminal deletion constructs (pcDNA4-△183-249 NDUFV2 and pcDNA4-△198-249 NDUFV2).

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. For generating constructs coding for fragments of pUL50 ( i.e. encoded amino acids 1–358, 1–205, 236–358, or 10–169) fused to GFP and β-gal, PCR amplification was performed as described above.

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy). .. In this vector cN-II ORF is placed under control of the inducible Gal1 promoter which allows the expression of the protein only when galactose is added to the growth medium.

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. For expression in S. aureus , edin was cloned using BamHI–PstI into pMK4-pPROT ( ) after PCR amplification using oligonucleotides E1r, 5′-CCCGGATCCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTGCAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′.

    Article Title: BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei
    Article Snippet: Vectors Mammalian expression vectors. .. The BILBO1 ORF and truncations were cloned into the pcDNA3 between HindIII-XbaI sites for BILBO1 full length and EcoRI-XhoI for the truncations or into pcDNA3.1 CT-GFP TOPO (Invitrogen).

    Modification:

    Article Title: BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei
    Article Snippet: The BILBO1 ORF and truncations were cloned into the pcDNA3 between HindIII-XbaI sites for BILBO1 full length and EcoRI-XhoI for the truncations or into pcDNA3.1 CT-GFP TOPO (Invitrogen). .. The pLew100X-3myc has been modified in the laboratory from pLew100 [ ].

    Transformation Assay:

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. Recombined plasmids were extracted from S. cerevisiae and transformed into E. coli DH5α for amplification.

    Article Title: Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold
    Article Snippet: The obtained PCR product was digested with EcoRI-XhoI and cloned into pYD1 vector (Invitrogen) digested with EcoRI-XhoI generating pYD1/FL- iafgp . .. The plasmids pYD1/FL- iafgp or pYD1 (without insert) were transformed into EBY100 competent cells, and cold tolerance assays were performed as mentioned in the Supplemental Methods.

    Derivative Assay:

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: The derived plasmid was used as the template for amplification by polymerase chain reaction (PCR) using Pfu DNA polymerase. .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector.

    Transfection:

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter. .. The MATCHMAKER two-hybrid system 3 (Clontech) was used according to the supplier’s protocol with the Sox6 bait plasmid, detailed above.

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: Paragraph title: 4.1. Cell Culture, Plasmids, and Transfection ... The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen).

    Ligation:

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: .. The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. To construct HA-tagged torsinA and torsinAΔE vectors, a single HA tag was introduced at the C terminus of torsinA and torsinAΔE by in vivo recombination in S. cerevisiae following a previously published protocol ( ).

    Cell Culture:

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: Paragraph title: 4.1. Cell Culture, Plasmids, and Transfection ... The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen).

    Generated:

    Article Title: Altered stoichiometry and nuclear delocalization of NonO and PSF promote cellular senescence
    Article Snippet: .. Plasmids pCR3.1-HA-NonO was constructed by excising full-length HA-NonO from pBS-HA-NonO1.4 vector with EcoRI/XhoI, and ligating it to EcoRI/XhoI digested pCR3.1 (Invitrogen). pBI-NonO2.4 was generated by excising full length NonO from p5.7NonO with XhoI and ligating it to SalI digested pBI (Clontech). .. GFP-NonO and deletion mutants were created by cloning a NonO cDNA into the pEGFP-C1 vector (Clontech).

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: DNA fragments for plasmid constructs were generated by PCR or RT–PCR and confirmed by sequence analysis. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: N-terminal deletion ( i.e. encoded amino acids 5–397, 10–397, or 15–397) and C-terminal deletion mutants of pUL50 ( i.e. encoded amino acids 1–181, 1–150, 1–130, 1–100, or 1–70) were generated by cloning of PCR products. .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP).

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. Recombinant bacterial strains were generated from an EDIN-producing human pathogenic strain of S. aureus (labeled S25) isolated from a 75-yr-old man with a spondylodiscitis-associated bacteremia (P. Boquet and P. Dellamonica, Hôpital ARCHET II, Nice, France).

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively. .. HSP27 mutants were generated by the insertion of oligonucleotides (Supplemental Table S2A).

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: Cells were maintained at 37 °C in a humidified atmosphere of 95% air/5% CO2 . pcDNA3.1-v5-hYSK1 was generated by PCR using the human cDNA clone- hYSK1 (Origene Technologies, Rockville, MD, USA) as a template. .. The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen).

    DNA Sequencing:

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct. .. Both AαQ and AβQ constructs were verified by DNA sequencing.

    Sequencing:

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. Briefly, primers LZJB12 and -13, encoding the HA tag sequence , were annealed and co-transformed into S. cerevisiae together with NotI-digested pRS426GPD-torsinA or torsinAΔE.

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The second recombinant PCR was performed to link the resultant Aβq with a 219 bp of chimeric cDNA that was PCR amplified from pRmHA3-Aβq-leuZ-bio-flag and consisted of a leucine zipper peptide, a flexible linker (RGGASGG), a biotinylation sequence and a flag-tag sequence. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: The resultant fragment was then cloned into the pGEM-T vector (Promega, Madison, WI, USA) and the sequence was confirmed by sequencing. .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector.

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: DNA fragments for plasmid constructs were generated by PCR or RT–PCR and confirmed by sequence analysis. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. In addition, expression constructs coding for mutant pUL50 carrying amino acid exchanges to alanine (single mutants D10A, L11A, V12A, Q13A, T15A, I18A, K20A, E56A, Y57A, N76A, G78A, P90A, L116A, K123A, R136A, G152A, and P153A; double mutants D10A/Q13A and L11A/V12A) were generated by site-directed mutagenesis using pcDNA-UL50-HA ( ) as template and oligonucleotide primers with nucleotides differing from the wild-type sequence ( ).

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: In bold the first triplet of cN-II ORF, boxed bases, spanning from −6 to +4, were designed to fit the Kozak consensus sequence ( http://en.wikipedia.org/wiki/Kozak_consensus_sequence ). .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy).

    Recombinant:

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The second recombinant PCR was performed to link the resultant Aβq with a 219 bp of chimeric cDNA that was PCR amplified from pRmHA3-Aβq-leuZ-bio-flag and consisted of a leucine zipper peptide, a flexible linker (RGGASGG), a biotinylation sequence and a flag-tag sequence. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: Plasmids were constructed for the recombinant expression of viral or cellular proteins in human cells. .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP).

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. Recombinant bacterial strains were generated from an EDIN-producing human pathogenic strain of S. aureus (labeled S25) isolated from a 75-yr-old man with a spondylodiscitis-associated bacteremia (P. Boquet and P. Dellamonica, Hôpital ARCHET II, Nice, France).

    In Vivo:

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. To construct HA-tagged torsinA and torsinAΔE vectors, a single HA tag was introduced at the C terminus of torsinA and torsinAΔE by in vivo recombination in S. cerevisiae following a previously published protocol ( ).

    Mutagenesis:

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. In addition, expression constructs coding for mutant pUL50 carrying amino acid exchanges to alanine (single mutants D10A, L11A, V12A, Q13A, T15A, I18A, K20A, E56A, Y57A, N76A, G78A, P90A, L116A, K123A, R136A, G152A, and P153A; double mutants D10A/Q13A and L11A/V12A) were generated by site-directed mutagenesis using pcDNA-UL50-HA ( ) as template and oligonucleotide primers with nucleotides differing from the wild-type sequence ( ).

    Article Title: BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei
    Article Snippet: The BILBO1 ORF and truncations were cloned into the pcDNA3 between HindIII-XbaI sites for BILBO1 full length and EcoRI-XhoI for the truncations or into pcDNA3.1 CT-GFP TOPO (Invitrogen). .. Mutations of the EF-hand domain 1 (D194A (GAT/GcT); N198A (AAC/gcC); D202A (GAC/GcC); D205A (GAC/GcC), the EF-hand domain 2 (D230A (GAC/GcC); N232A (AAC/gcC); E241A (GAA/GcA), and the serine 163 mutations (S163D (TCG/gat) and S163A (TCG/gCG) were done by site-directed mutagenesis following the instructions from the Agilent QuickChange Site-directed Mutagenesis kit.

    Isolation:

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. Recombinant bacterial strains were generated from an EDIN-producing human pathogenic strain of S. aureus (labeled S25) isolated from a 75-yr-old man with a spondylodiscitis-associated bacteremia (P. Boquet and P. Dellamonica, Hôpital ARCHET II, Nice, France).

    Labeling:

    Article Title: Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
    Article Snippet: DNA constructs and bacterial genetic The DNA encoding EDIN (NCBI M63917) was cloned using EcoRI–XhoI into pcDNA4 (Invitrogen) after PCR amplification on the E1 strain of S. aureus (provided by M. Sugai, Hiroshima University, Hiroshima, Japan; ) using the following oligonucleotides: E1r, 5′-CCCGAATTCATGAAAAACAAATTACTTTTTAAAATTTTTTTG-3′ and E1f, 5′-CCCCTCGAGCTATTTTTTAAAAACAATAGCTGTTATTATG-3′. .. Recombinant bacterial strains were generated from an EDIN-producing human pathogenic strain of S. aureus (labeled S25) isolated from a 75-yr-old man with a spondylodiscitis-associated bacteremia (P. Boquet and P. Dellamonica, Hôpital ARCHET II, Nice, France).

    Purification:

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector. .. Construction of plasmids expressing truncated NDUFV2 proteins The pcDNA4-NDUFV2 vector was used as the template for generation of its N-terminal deletion constructs (pcDNA4-△1-18 NDUFV2, pcDNA4-△1-32 NDUFV2 and pcDNA4-△1-50 NDUFV2) and C-terminal deletion constructs (pcDNA4-△183-249 NDUFV2 and pcDNA4-△198-249 NDUFV2).

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: .. The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen). .. The GST-hYSK1 was inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, Buckinghamshire, UK).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To generate the plasmid pGADT7 (Clontech) for the Co-IP, the amplified PCR fragment encoding the first 108 amino acids of the Prtb gene (GenBank accession no. ), and RT–PCR of the entire gene (168 amino acids), were cloned in-frame into EcoRI–XhoI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: MAPK4, PTEN, and HSP27 cDNAs were cloned by RT–PCR from U2OS cells. .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively.

    Quantitative RT-PCR:

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: Creation of the human Sp1 construct with HA-tag added to the N terminus was accomplished via real-time RT-PCR amplification of human PBMC cDNA with the following primers: Sp1 forward, 5′-CGCGAATTCATGAGCGACCAAGATCACTCCATGGA-3′; Sp1 reverse, 5′-CGCCTCGAGTCAGAAGCCATTGCCACTGATATTAATGGAC-3′. .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen).

    Agarose Gel Electrophoresis:

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: A construct pMT-H-2aα/Eαk , plasmid (kindly provided by Dr. Dario Vignali from St. Jude Childrens’ research hospital, Memphis, TN) was digested with EcoRI/AscI and separated on a 1 % agarose gel. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Plasmid Preparation:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: .. Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. For the construction of the mammalian and bacterial expression vectors, the gateway cloning system technology (Thermo Fischer Scientific, USA) was applied.

    Article Title: The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia *
    Article Snippet: .. The torsinA and torsinAΔE open reading frames (ORF) were subcloned into the yeast expression vector pRS426GPD ( ) by double restriction enzyme digestion with EcoRI/XhoI (Fermentas, Thermo Scientific) of pcDNA3.1-torsinA and pcDNA3.1-torsinAΔE and ligation into EcoRI/XhoI-linearized pRS426GPD ( B ). .. To construct HA-tagged torsinA and torsinAΔE vectors, a single HA tag was introduced at the C terminus of torsinA and torsinAΔE by in vivo recombination in S. cerevisiae following a previously published protocol ( ).

    Article Title: Altered stoichiometry and nuclear delocalization of NonO and PSF promote cellular senescence
    Article Snippet: .. Plasmids pCR3.1-HA-NonO was constructed by excising full-length HA-NonO from pBS-HA-NonO1.4 vector with EcoRI/XhoI, and ligating it to EcoRI/XhoI digested pCR3.1 (Invitrogen). pBI-NonO2.4 was generated by excising full length NonO from p5.7NonO with XhoI and ligating it to SalI digested pBI (Clontech). .. GFP-NonO and deletion mutants were created by cloning a NonO cDNA into the pEGFP-C1 vector (Clontech).

    Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis
    Article Snippet: .. Reporter vectors The parental vector pUASt-XBP1-EGFP was a kind gift from H Steller and HD Ryoo (NYU School of Medicine, USA) . pMT-V5-eroGFP was obtained by cloning a EcoRI/XhoI -digested fragment encompassing the coding fragment of roGFP-HDEL from the p28M kar2ss-roGFP2-HDEL vector into the pMT-His-V5 expression vector (Invitrogen). .. The inducible IRE1-EGFP construct pMT-IRE1-Flag-EGFP ( ) was obtained by cloning a PCR product from pcDNA5.1/3xFLAGhIRE1-EGFP with flanking KpnI and NotI sites in the pMT-His-V5 vector.

    Article Title: Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold
    Article Snippet: .. The obtained PCR product was digested with EcoRI-XhoI and cloned into pYD1 vector (Invitrogen) digested with EcoRI-XhoI generating pYD1/FL- iafgp . .. The plasmids pYD1/FL- iafgp or pYD1 (without insert) were transformed into EBY100 competent cells, and cold tolerance assays were performed as mentioned in the Supplemental Methods.

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: The amplified PCR product was digested with XhoI/HindIII and cloned into pPicaGene-Basic vector II (Toyo-ink), which yielded TBX21 -Luc. .. The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen).

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct. .. Both AαQ and AβQ constructs were verified by DNA sequencing.

    Article Title: Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy
    Article Snippet: .. The resulting plasmid was digested with EcoRI/XhoI, and the DNA fragment containing the desired cDNA was then purified and ligated with the pcDNA4/TO/myc -His A vector (Invitrogen) using the same restriction sites to generate the pcDNA4-NDUFV2 expressing vector. .. Construction of plasmids expressing truncated NDUFV2 proteins The pcDNA4-NDUFV2 vector was used as the template for generation of its N-terminal deletion constructs (pcDNA4-△1-18 NDUFV2, pcDNA4-△1-32 NDUFV2 and pcDNA4-△1-50 NDUFV2) and C-terminal deletion constructs (pcDNA4-△183-249 NDUFV2 and pcDNA4-△198-249 NDUFV2).

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter. .. The MATCHMAKER two-hybrid system 3 (Clontech) was used according to the supplier’s protocol with the Sox6 bait plasmid, detailed above.

    Article Title: Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53 *
    Article Snippet: .. After cleavage with EcoRI/XhoI or HindIII/BamHI, respectively, PCR products were inserted into the vector pcDNA3.1 (Invitrogen), resulting in expression constructs coding for N-terminal deletion mutants of pUL50 fused C-terminally to a hemagglutinin (HA) tag or inserted into the vector peGPF-N1 (Clontech) resulting in expression constructs coding for C-terminal deletion mutants of pUL50 fused C-terminally to the green fluorescent protein (GFP). .. For generating constructs coding for fragments of pUL50 ( i.e. encoded amino acids 1–358, 1–205, 236–358, or 10–169) fused to GFP and β-gal, PCR amplification was performed as described above.

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy). .. In this vector cN-II ORF is placed under control of the inducible Gal1 promoter which allows the expression of the protein only when galactose is added to the growth medium.

    Article Title: BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei
    Article Snippet: The BILBO1 ORF and truncations were cloned into the pcDNA3 between HindIII-XbaI sites for BILBO1 full length and EcoRI-XhoI for the truncations or into pcDNA3.1 CT-GFP TOPO (Invitrogen). .. Trypanosome expression vector.

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively. .. HSP27 mutants were generated by the insertion of oligonucleotides (Supplemental Table S2A).

    Article Title: p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions
    Article Snippet: The PCR product was purified, digested with EcoRI/XhoI , and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen). .. The GST-hYSK1 was inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, Buckinghamshire, UK).

    Co-Immunoprecipitation Assay:

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To generate the plasmid pGADT7 (Clontech) for the Co-IP, the amplified PCR fragment encoding the first 108 amino acids of the Prtb gene (GenBank accession no. ), and RT–PCR of the entire gene (168 amino acids), were cloned in-frame into EcoRI–XhoI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    Positron Emission Tomography:

    Article Title: Expression of Bovine Cytosolic 5?-Nucleotidase (cN-II) in Yeast: Nucleotide Pools Disturbance and Its Consequences on Growth and Homologous Recombination
    Article Snippet: Plasmid and Yeast Strain The cN-II cDNA was amplified by PCR using pET-28c+cNII as template. .. PCR product was EcoRI-XhoI digested and cloned into the pYES2 plasmid (Life Technologies, Italy).

    Two Hybrid Screening:

    Article Title: Sox6 regulation of cardiac myocyte development
    Article Snippet: To construct the plasmid pGBKT7 (Clontech) as bait for the yeast two-hybrid screen and for the co-immunoprecipitation (Co-IP), an amplified PCR fragment encoding amino acids 139–304 (including the coiled-coil domain) of mouse Sox6 (GenBank accession no. ) was cloned in-frame into EcoRI–SalI sites. .. For transient transfection, complete cDNAs of the mouse Sox6 and Prtb genes were cloned into EcoRI, and EcoRI–XhoI, respectively, to pCDNA3.1/Zeo vector (Invitrogen) driven by the CMV promoter.

    FLAG-tag:

    Article Title: Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4
    Article Snippet: Expression vectors and cloning For the Y2H screen, the N-terminal kinase domain of RIPK4 (1-340) was subcloned from pCR3 VSV-N-RIPK4 (Meylan et al., 2002) into Gal4-DNA-BD containing pGBKT7 (Clontech, USA) yeast expression plasmid by using EcoRI-XhoI and EcoRI-SalI (Invitrogen, USA) restriction enzymes, respectively. .. Amplified RIPK4 and K51R ORFs were cloned into the p3xFlag CMV/DEST expression vector (Invitrogen, USA) to provide expression of RIPK4 and K51R in mammalian cells in fusion with the N-terminal Flag tag.

    Article Title: HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells
    Article Snippet: The amplified fragment was digested with EcoRI/XhoI and subcloned into HA-tagged pcDNA3 (Invitrogen). .. Tax construct with FLAG-tag added to the N terminus was prepared via PCR amplification of template DNA ( ) with the following primers: Tax forward, 5′-CGCGAATTCATGGCCCACTTCCCAGGGTTT-3′; Tax reverse, 5′-CGCCTCGAGTCAGACTTCTGTTTCACGGAAATGTTTTTC-3′.

    Article Title: Peptide Ligand Structure and I-Aq Binding Avidity Influence T Cell Signaling Pathway Utilization
    Article Snippet: The second recombinant PCR was performed to link the resultant Aβq with a 219 bp of chimeric cDNA that was PCR amplified from pRmHA3-Aβq-leuZ-bio-flag and consisted of a leucine zipper peptide, a flexible linker (RGGASGG), a biotinylation sequence and a flag-tag sequence. .. The resultant chimeric AβQ:LeuZ:biotin:flag cDNA was then digested with EcoRI/XhoI and subcloned into the same sites of pMT-V5 vector (InVitrogen, Carlsbad, CA) to product pMT-AβQ-LeuZ-bio-flag construct.

    Article Title: IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling
    Article Snippet: .. PCR products were subcloned in Zero-Blunt plasmid (Invitrogen) or pGEM-T vector (Promega) and sequenced before insertion via EcoRI/XhoI at the 3′ end of a firefly luciferase, Flag tag in pcDNA3.1 (Invitrogen), pEGFP-C2 vector (Clontech), or mRFP-C1 respectively. .. HSP27 mutants were generated by the insertion of oligonucleotides (Supplemental Table S2A).

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