bamhi xhoi  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bamhi xhoi
    Bamhi Xhoi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamhi xhoi/product/Thermo Fisher
    Average 96 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    bamhi xhoi - by Bioz Stars, 2020-04
    96/100 stars

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    Related Articles

    Transduction:

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Clone Assay:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing. .. Jurkat cells were transfected using the nucleofector 4D machine and stable clones selected using G418 (Gibco).

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5. .. The PCR product generated with oligonucleotides 5′-CCGGATCCGAATGGCCAGCGTCCAGGAG-3′ and 5′-GGCTCGAGTGCCGTCTTCCCAGCAAAAG-3′ was digested with BamHI-XhoI and cloned into BamHI-XhoI sites of pACTII to generate pACTII-Apc5Δ2 (N-terminal part, residues 1 to 395).

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Article Title: Identification of CELF splicing activation and repression domains in vivo
    Article Snippet: .. Plasmids CELF4 and ETR-3 deletion mutants truncated at the positions indicated in – were generated by PCR and cloned using BamHI/XhoI into pcDNA3.1HisC(+) (Invitrogen). .. The chicken cTNT minigene plasmid RTB33.51 has been described previously ( ).

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: .. Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. In both KO constructs, flanking regions were cloned upstream and downstream of the nptII gene driven by a 35S promoter (for geneticin resistance).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: .. The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Amplification:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: Plasmid and siRNAs construction To generate vmiRNA expression constructs, the candidate vmiRNA hairpin domains with ~100-bp flanking sequences were amplified from LAI DNA. .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen).

    Article Title: Hypoxia-Response Element (HRE)-Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium
    Article Snippet: .. The cDNA fragments of wild-type HIF-1α, DN-HIF-1α, and wild-type HIF-1β with the full coding region were amplified by RT-PCR and inserted into the BamHI-XhoI, HindIII-XhoI, and BamHI-XhoI sites of pcDNA3.1/v5-his expression plasmid, respectively, (Invitrogen) to create expression constructs of pcDNA3.1-HIF-1α, pcDNA3.1-DN-HIF-1α, and pcDNA3.1-HIF-1β as described ( ). .. Cell transfection and assays for reporter gene products were carried out as described previously ( ).

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: PCR amplification of 5′ and 3′ flanking regions (941 bp and 932 bp, respectively) of the PpATG7 gene was performed using primers p17/p18 and p19/p20 shown in Table S1. .. Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid.

    Construct:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: Paragraph title: Plasmids and Constructs ... The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen).

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5. .. The SmaI-XhoI fragment of pGAD-DH#134 was inserted into the corresponding sites of pACTII to construct pACTII-Apc5Δ1 (C-terminal part, residues 395 to 755). pFlag-Apc5, containing the T7 promoter-driven Apc5 coding region fused to Flag tag at its N terminus, was a kind gift from C. Hoog. pACTII-IRP, expressing the GAL4 activation domain fused to the iron-responsive element binding protein (IRP), was kindly provided by M. Wickens ( ). pACTII-hPABP, harboring the human PABP1 fused to GAL4 activation domain, was constructed by generating a PCR product using oligonucleotides 5′-GGGGATCCAGATGAACCCCAGTGCC-3′ and 5′-GGGGATCCTTCGGTGAAGCACAAG-3′, with pGEX2T-PABP ( ) as template.

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: Plasmid and siRNAs construction To generate vmiRNA expression constructs, the candidate vmiRNA hairpin domains with ~100-bp flanking sequences were amplified from LAI DNA. .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen).

    Article Title: Hypoxia-Response Element (HRE)-Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium
    Article Snippet: .. The cDNA fragments of wild-type HIF-1α, DN-HIF-1α, and wild-type HIF-1β with the full coding region were amplified by RT-PCR and inserted into the BamHI-XhoI, HindIII-XhoI, and BamHI-XhoI sites of pcDNA3.1/v5-his expression plasmid, respectively, (Invitrogen) to create expression constructs of pcDNA3.1-HIF-1α, pcDNA3.1-DN-HIF-1α, and pcDNA3.1-HIF-1β as described ( ). .. Cell transfection and assays for reporter gene products were carried out as described previously ( ).

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. In both KO constructs, flanking regions were cloned upstream and downstream of the nptII gene driven by a 35S promoter (for geneticin resistance).

    Article Title: Phosphorylation of I?B? at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells *
    Article Snippet: Plasmid V5-TOPK was constructed. .. Sequentially, PCR was performed with the cDNA using sense strand, 5′-CGCGGATCCCGATGGAAGGGATCAGTAATTT-3′ harboring the BamHI site and antisense strand 5′-CCGCTCGAGCGGGACATCTGTTTCCAGAGCTT-3′ harboring the XhoI site, and the PCR product cut with BamHI/XhoI was inserted into the BamHI/XhoI sites of pcDNA6/V5-His A (Invitrogen).

    Luciferase:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: Phosphorylation of I?B? at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells *
    Article Snippet: Sequentially, PCR was performed with the cDNA using sense strand, 5′-CGCGGATCCCGATGGAAGGGATCAGTAATTT-3′ harboring the BamHI site and antisense strand 5′-CCGCTCGAGCGGGACATCTGTTTCCAGAGCTT-3′ harboring the XhoI site, and the PCR product cut with BamHI/XhoI was inserted into the BamHI/XhoI sites of pcDNA6/V5-His A (Invitrogen). .. Plasmids, cIAP2 promoter-driven luciferase reporter vectors, wild type, and NF-κB mutants, were kindly provided by Dr. Hinrich Gronemeyer ( ).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Influenza pseudotyped lentivirus particles expressing the firefly luciferase reporter gene were generated by plasmid co-transfection as previously described [ ] with the following variations.

    Expressing:

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: .. Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing. .. Jurkat cells were transfected using the nucleofector 4D machine and stable clones selected using G418 (Gibco).

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: .. The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen). .. To create a full-length pET-NPDC-1 bacterial expression construct, the pGEM-NPDC-1 construct was digested with AvrII , filled-in with Klenow and then digested with NotI .

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5. .. The SmaI-XhoI fragment of pGAD-DH#134 was inserted into the corresponding sites of pACTII to construct pACTII-Apc5Δ1 (C-terminal part, residues 395 to 755). pFlag-Apc5, containing the T7 promoter-driven Apc5 coding region fused to Flag tag at its N terminus, was a kind gift from C. Hoog. pACTII-IRP, expressing the GAL4 activation domain fused to the iron-responsive element binding protein (IRP), was kindly provided by M. Wickens ( ). pACTII-hPABP, harboring the human PABP1 fused to GAL4 activation domain, was constructed by generating a PCR product using oligonucleotides 5′-GGGGATCCAGATGAACCCCAGTGCC-3′ and 5′-GGGGATCCTTCGGTGAAGCACAAG-3′, with pGEX2T-PABP ( ) as template.

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: Plasmid and siRNAs construction To generate vmiRNA expression constructs, the candidate vmiRNA hairpin domains with ~100-bp flanking sequences were amplified from LAI DNA. .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen).

    Article Title: Hypoxia-Response Element (HRE)-Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium
    Article Snippet: .. The cDNA fragments of wild-type HIF-1α, DN-HIF-1α, and wild-type HIF-1β with the full coding region were amplified by RT-PCR and inserted into the BamHI-XhoI, HindIII-XhoI, and BamHI-XhoI sites of pcDNA3.1/v5-his expression plasmid, respectively, (Invitrogen) to create expression constructs of pcDNA3.1-HIF-1α, pcDNA3.1-DN-HIF-1α, and pcDNA3.1-HIF-1β as described ( ). .. Cell transfection and assays for reporter gene products were carried out as described previously ( ).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: .. The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Modification:

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Transformation Assay:

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Over Expression:

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: .. Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Derivative Assay:

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Hybridization:

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: Complementary DNA oligomers consisting of human pre-miR-150, flanked by 30 additional nucleotides from the pri-miRNA sequence and BamHI/XhoI sticky ends, were synthesised (Eurofins MWG Operon) and hybridised together in hybridisation buffer (10 mM TrisHCl, 100 mM NaCl, 1 mM EDTA). .. Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing.

    Transfection:

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing. .. Jurkat cells were transfected using the nucleofector 4D machine and stable clones selected using G418 (Gibco).

    Article Title: Phosphorylation of I?B? at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells *
    Article Snippet: Paragraph title: Plasmids and Transfection ... Sequentially, PCR was performed with the cDNA using sense strand, 5′-CGCGGATCCCGATGGAAGGGATCAGTAATTT-3′ harboring the BamHI site and antisense strand 5′-CCGCTCGAGCGGGACATCTGTTTCCAGAGCTT-3′ harboring the XhoI site, and the PCR product cut with BamHI/XhoI was inserted into the BamHI/XhoI sites of pcDNA6/V5-His A (Invitrogen).

    Sequencing:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: The NLS used for the Luc -NLS construct was already described ( ) and Luc -NLS was generated by inserting the NLS sequence within the ApaI/BamHI site of pHRLuc-C3 (Perkin Elmer Life Sciences). .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen).

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: .. Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing. .. Jurkat cells were transfected using the nucleofector 4D machine and stable clones selected using G418 (Gibco).

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: .. Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. Positive clones were selected by colony PCR and sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Perkin Elmer Applied Biosystem).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Ligation:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: .. Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. In both KO constructs, flanking regions were cloned upstream and downstream of the nptII gene driven by a 35S promoter (for geneticin resistance).

    Generated:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: The NLS used for the Luc -NLS construct was already described ( ) and Luc -NLS was generated by inserting the NLS sequence within the ApaI/BamHI site of pHRLuc-C3 (Perkin Elmer Life Sciences). .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen).

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: The complete coding region of human Apc5 was generated by reverse transcription-PCR (RT-PCR) using oligonucleotides 5′-GGGGATCCGCTAGCGCCATGGCCAGCGTCC-3′ and 5′-CCGCTCGAGGGGATGTCCTCTAGAG-3′ and the Marathon kit (from human testis; Clontech) as a template. .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5.

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Article Title: Identification of CELF splicing activation and repression domains in vivo
    Article Snippet: .. Plasmids CELF4 and ETR-3 deletion mutants truncated at the positions indicated in – were generated by PCR and cloned using BamHI/XhoI into pcDNA3.1HisC(+) (Invitrogen). .. The chicken cTNT minigene plasmid RTB33.51 has been described previously ( ).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Influenza pseudotyped lentivirus particles expressing the firefly luciferase reporter gene were generated by plasmid co-transfection as previously described [ ] with the following variations.

    Polymerase Chain Reaction:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: .. The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen). .. To create a full-length pET-NPDC-1 bacterial expression construct, the pGEM-NPDC-1 construct was digested with AvrII , filled-in with Klenow and then digested with NotI .

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5. .. The same PCR product was also digested with NheI-XhoI and inserted into the NheI-XhoI sites of pET28b (Novagen) to generate pET28b-Apc5.

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: Primers used for PCR were shown in Table S1. cDNAs encoding human Miro1, its splicing variants, Miro2, TRAK1, and TRAK2 were obtained by RT-PCR using total RNA from HEK cells. .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants.

    Article Title: Identification of CELF splicing activation and repression domains in vivo
    Article Snippet: .. Plasmids CELF4 and ETR-3 deletion mutants truncated at the positions indicated in – were generated by PCR and cloned using BamHI/XhoI into pcDNA3.1HisC(+) (Invitrogen). .. The chicken cTNT minigene plasmid RTB33.51 has been described previously ( ).

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: PCR amplification of 5′ and 3′ flanking regions (941 bp and 932 bp, respectively) of the PpATG7 gene was performed using primers p17/p18 and p19/p20 shown in Table S1. .. Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid.

    Article Title: Phosphorylation of I?B? at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells *
    Article Snippet: .. Sequentially, PCR was performed with the cDNA using sense strand, 5′-CGCGGATCCCGATGGAAGGGATCAGTAATTT-3′ harboring the BamHI site and antisense strand 5′-CCGCTCGAGCGGGACATCTGTTTCCAGAGCTT-3′ harboring the XhoI site, and the PCR product cut with BamHI/XhoI was inserted into the BamHI/XhoI sites of pcDNA6/V5-His A (Invitrogen). .. Plasmids, cIAP2 promoter-driven luciferase reporter vectors, wild type, and NF-κB mutants, were kindly provided by Dr. Hinrich Gronemeyer ( ).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: .. The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Binding Assay:

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: The SmaI-XhoI Apc5 fragment of pGAD-GH#134 was fused to GAL4 DNA binding domain by its insertion into the SmaI-SalI sites of pGBT9 (Clontech) to create pGBT-hApc5(395-755). .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5.

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Mutagenesis:

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: .. Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Purification:

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: The complete coding region of human Apc5 was generated by reverse transcription-PCR (RT-PCR) using oligonucleotides 5′-GGGGATCCGCTAGCGCCATGGCCAGCGTCC-3′ and 5′-CCGCTCGAGGGGATGTCCTCTAGAG-3′ and the Marathon kit (from human testis; Clontech) as a template. .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5.

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: Primers used for PCR were shown in Table S1. cDNAs encoding human Miro1, its splicing variants, Miro2, TRAK1, and TRAK2 were obtained by RT-PCR using total RNA from HEK cells. .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants.

    Article Title: Hypoxia-Response Element (HRE)-Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium
    Article Snippet: .. The cDNA fragments of wild-type HIF-1α, DN-HIF-1α, and wild-type HIF-1β with the full coding region were amplified by RT-PCR and inserted into the BamHI-XhoI, HindIII-XhoI, and BamHI-XhoI sites of pcDNA3.1/v5-his expression plasmid, respectively, (Invitrogen) to create expression constructs of pcDNA3.1-HIF-1α, pcDNA3.1-DN-HIF-1α, and pcDNA3.1-HIF-1β as described ( ). .. Cell transfection and assays for reporter gene products were carried out as described previously ( ).

    Positron Emission Tomography:

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen). .. To create a full-length pET-NPDC-1 bacterial expression construct, the pGEM-NPDC-1 construct was digested with AvrII , filled-in with Klenow and then digested with NotI .

    Cotransfection:

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Influenza pseudotyped lentivirus particles expressing the firefly luciferase reporter gene were generated by plasmid co-transfection as previously described [ ] with the following variations.

    cDNA Library Assay:

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: pGAD-GH#134 containing residues 395 to 755 of human Apc5 fused to the GAL4 activation domain, was selected from the previously described HeLa cDNA library ( ). .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5.

    Activated Clotting Time Assay:

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: To generate the pcDNA3.1-aRARγ and pcDNA3.1-HA-aRARγ mammalian expression constructs for axolotl RAR gamma, and the pGEX-aRARγ and pET32-aRARγ bacterial expression constructs for axolotl RAR gamma, we used the pFLAG-aRARγ construct as template in a PCR reaction with the following primers: forward 5′-AAG GAT CCA TGT ACG ACT GCA TGG AGG CC-3′ and reverse 5′-AAC TCG AGC TAG AGC TCT TTG GAA CT-3′. .. The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen).

    Plasmid Preparation:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: The myc-tagged SUMO1 construct used was subcloned as an XhoI fragment in the XhoI site of a myc-tagged version of pcDNA3.1 vector. .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen).

    Article Title: CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells
    Article Snippet: .. Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas), and verified by sequencing. .. Jurkat cells were transfected using the nucleofector 4D machine and stable clones selected using G418 (Gibco).

    Article Title: ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING
    Article Snippet: The PCR fragment was digested with BamHI/XhoI and subloned into the Bam HI /Xho I sites of the mammalian expression vectors pcDNA3.1/Zeo (Invitrogen) and pcDNA3.1/Zeo/3HA (a gift from Dr. Douglas Andres at the University of Kentucky), and the bacterial expression vectors pGEX-KG (Amersham) and pET32a (Novagen). .. This fragment was subcloned into the Eco RV /Not I sites of the bacterial protein expression vector pET32c (Novagen).

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: .. Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Article Title: New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes
    Article Snippet: .. Amplified cDNAs each coding for full-length Miro1 splicing variants, Miro1-var2ΔTMD (residues at 2–624 aa), Miro1-var4ΔTMD (residues at 2–665 aa), and full-length Miro2 were cloned into the BamHI–XhoI sites in a pcDNA3.1Zeo+ /HA2 -Ub vector ( ) by replacing the ubiquitin-encoding fragment, generating pcDNA3.1Zeo-based plasmids encoding N-terminally HA2 -tagged Miro1 variants and full-length Miro2. cDNA encoding TRAK1 and TRAK2 were likewise cloned into pcDNA3.1Zeo+ /FLAG-Ub vector and pcDNA3.1Zeo+ /HA2 -Ub vector ( ) at the BamHI–XhoI and BamHI–NotI sites, respectively, generating pcDNA3.1Zeo/FLAG-TRAK1, pcDNA3.1Zeo/FLAG-TRAK2, and pcDNA3.1Zeo/HA2 -TRAK2. cDNAs encoding Miro1 splicing variants were also ligated into the BamHI–XhoI sites in a modified pcDNA3.1Zeo+ vector (Invitrogen) encoding an N-terminal mCherry tag derived from an mCherry-C1 vector (Takara Bio Inc.), generating pcDNA3.1Zeo vector–encoding mCherry-Miro1 variants. .. The nontagged version of Miro1 variants was generated by cloning cDNAs of full-length Miro1 variants into the BamHI–XhoI sites in a pcDNA3.1Zeo+ vector.

    Article Title: Identification of CELF splicing activation and repression domains in vivo
    Article Snippet: Plasmids CELF4 and ETR-3 deletion mutants truncated at the positions indicated in – were generated by PCR and cloned using BamHI/XhoI into pcDNA3.1HisC(+) (Invitrogen). .. The chicken cTNT minigene plasmid RTB33.51 has been described previously ( ).

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Article Title: Hypoxia-Response Element (HRE)-Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium
    Article Snippet: .. The cDNA fragments of wild-type HIF-1α, DN-HIF-1α, and wild-type HIF-1β with the full coding region were amplified by RT-PCR and inserted into the BamHI-XhoI, HindIII-XhoI, and BamHI-XhoI sites of pcDNA3.1/v5-his expression plasmid, respectively, (Invitrogen) to create expression constructs of pcDNA3.1-HIF-1α, pcDNA3.1-DN-HIF-1α, and pcDNA3.1-HIF-1β as described ( ). .. Cell transfection and assays for reporter gene products were carried out as described previously ( ).

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: .. Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. In both KO constructs, flanking regions were cloned upstream and downstream of the nptII gene driven by a 35S promoter (for geneticin resistance).

    Article Title: Phosphorylation of I?B? at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells *
    Article Snippet: Plasmid V5-TOPK was constructed. .. Sequentially, PCR was performed with the cDNA using sense strand, 5′-CGCGGATCCCGATGGAAGGGATCAGTAATTT-3′ harboring the BamHI site and antisense strand 5′-CCGCTCGAGCGGGACATCTGTTTCCAGAGCTT-3′ harboring the XhoI site, and the PCR product cut with BamHI/XhoI was inserted into the BamHI/XhoI sites of pcDNA6/V5-His A (Invitrogen).

    Article Title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus
    Article Snippet: .. The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid. .. Both gene sequences were confirmed by bidirectional custom sequencing (GATC Biotech) using primers binding to the arms of the pI.18 vector.

    Selection:

    Article Title: The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells
    Article Snippet: Overexpression of pitrilysin pcDNA3.1 vectors containing cDNAs of human pitrilysin (PITRM) [ ] or its inactive E107Q mutant [ ] (PITRMx) tagged with a flag sequence were digested with BamHI/XhoI and subcloned into the lentiviral vector pLenti6/V5 (Invitrogen). .. For selection of INS 832/13 stable cells overexpressing pitrilysin or inactive pitrilysin, cells were transduced with the appropriate lentivirus and then selected with 10 μg/ml blasticidin.

    Bioluminescence Resonance Energy Transfer:

    Article Title: Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation
    Article Snippet: .. To generate the other BRET constructs, Renilla Luciferase ( Luc ) or YFP were cloned together with the cDNA of interest by a three-piece ligation in the BamHI/XhoI or BamHI/XbaI site of pcDNA3.1(+) (Invitrogen). .. Luc was amplified by PCR from the pcDNA3-CXCR4-Luc vector ( ) and cloned either at the COOH-terminal of the cDNA of interest as an EcoRI-XhoI fragment (primers used: “ Luc _EcoRI_ATG_C-term sense” and “ Luc _Stop_XhoI_C-term antisense” primers) or at the N-terminal as a BamHI-EcoRI fragment (primers used: “ Luc _BamHI_ATG_N-term sense” and “ Luc _no-Stop_EcoRI_N-term antisense”).

    Knock-Out:

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. Overviews of PpATG5 , PpATG7 and the resulting knockout constructs are depicted in Figure S2A.

    Activation Assay:

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: pGAD-GH#134 containing residues 395 to 755 of human Apc5 fused to the GAL4 activation domain, was selected from the previously described HeLa cDNA library ( ). .. This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5.

    FLAG-tag:

    Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation
    Article Snippet: This PCR product was digested with BamHI-XhoI and inserted into BamHI-XhoI sites of pcDNA3 (Invitrogen) to generate pcDNA3-Apc5 and into BamHI and XhoI sites of pACTII (Clontech) to generate pACTII-Apc5. .. The SmaI-XhoI fragment of pGAD-DH#134 was inserted into the corresponding sites of pACTII to construct pACTII-Apc5Δ1 (C-terminal part, residues 395 to 755). pFlag-Apc5, containing the T7 promoter-driven Apc5 coding region fused to Flag tag at its N terminus, was a kind gift from C. Hoog. pACTII-IRP, expressing the GAL4 activation domain fused to the iron-responsive element binding protein (IRP), was kindly provided by M. Wickens ( ). pACTII-hPABP, harboring the human PABP1 fused to GAL4 activation domain, was constructed by generating a PCR product using oligonucleotides 5′-GGGGATCCAGATGAACCCCAGTGCC-3′ and 5′-GGGGATCCTTCGGTGAAGCACAAG-3′, with pGEX2T-PABP ( ) as template.

    Gel Extraction:

    Article Title: Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs
    Article Snippet: .. PCR products were excised from gel, purified with the QIAquick gel extraction kit (Qiagen), BamHI/XhoI digested and cloned in the pcDNA6.2-GW/EmGFP-miR vector (Invitrogen). .. These plasmids were transformed into bacterial TOP10 cells.

    Homologous Recombination:

    Article Title: Autophagy is required for gamete differentiation in the moss Physcomitrella patens
    Article Snippet: Fragments were digested with BamHI/XhoI and SpeI/NsiI respectively (Thermo Fisher Scientific, FD0055, FD0694, FD1254 and FD0734), and cloned by sequential sticky end ligation into the pMT164 plasmid. .. Flanking regions ensure specific targeting of the PpATG5 and PpATG7 genes and their exchange for the nptII gene in the genome by homologous recombination.

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    Thermo Fisher bamhi aid ecori linker hindiii aid e58a xhoi cassettes
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