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  • 90
    Thermo Fisher lr clonase ii plus
    FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite <t>Clonase</t> strategy utilizing modified MuLE entry vectors.
    Lr Clonase Ii Plus, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lr clonase mix
    Overview of the lentiviral (A) or retroviral (B) vectors for protein expression or depletion. Entry vectors can be recombined using the LR <t>clonase</t> into the Destination vectors connected by arrows. For RNAi-mediated depletion, miRNA-based and shRNA-based vectors are available for either constitutive or inducible depletions (see Figure 2A for details). For expression, various tags are available (see Figure 2B for details). The RNAi cassettes can be inserted in the middle of the lentiviral backbone (pLenti X1 series), in the 3′ LTR (pLenti X2 series) or in a backbone expressing GFP (pLenti CMV GFP). For RNAi studies using retroviruses, all the Destination cassettes have been inserted in the 3′ LTR of the vectors. For protein expression, the EF-1α, PGK or CMV promoters are available for lentiviruses. CMV-driven constructs can be either constitutive or inducible. For the retroviral vectors, no constitutive expression of cDNAs can be attained in T-REx cell lines using the pQCXI series vectors because they all contain the inducible CMV/TO promoter. Similarly, the drug resistance gene will be repressed in a T-REx cell line since it is after an IRES element under the control of the CMV/TO promoter. However, the pQCXP CMV/TO can be used for inducible expression of cDNAs under constitutive puromycin selection in T-REx cell lines. See Table 2 for details.
    Lr Clonase Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gateway lr clonase ii enzyme mix
    FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite <t>Clonase</t> strategy utilizing modified MuLE entry vectors.
    Gateway Lr Clonase Ii Enzyme Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lr clonase
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lr clonase ii reaction kit
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Ii Reaction Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lr clonase system
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gateway lr clonase reaction
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Gateway Lr Clonase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher gateway lr clonase reactions
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Gateway Lr Clonase Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher standard lr clonase
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Standard Lr Clonase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher lr clonase kit
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Thermo Fisher standard lr clonase reaction
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Standard Lr Clonase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher lr clonase catalysed reaction
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Catalysed Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lr clonase reaction kit
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Reaction Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher lr clonase enzyme kit
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Enzyme Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher lr clonease
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher lr clonase reaction mixture
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase Reaction Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Thermo Fisher multigateway lr clonase
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Multigateway Lr Clonase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher multiplex lr clonase reaction
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Multiplex Lr Clonase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher lr clonase i
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Lr Clonase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bp clonases
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Bp Clonases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fusion constructs lr clonase
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Fusion Constructs Lr Clonase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher attl attr lr clonase
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Attl Attr Lr Clonase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher multisite gateway three fragment lr clonase reaction
    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
    Gatewayr Lr Clonase Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP <t>Clonase</t> reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.
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    Image Search Results


    FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite Clonase strategy utilizing modified MuLE entry vectors.

    Journal: Gene

    Article Title: A Splice Junction-Targeted CRISPR Approach (spJCRISPR) Reveals Human FOXO3B To Be A Protein-Coding Gene

    doi: 10.1016/j.gene.2018.06.048

    Figure Lengend Snippet: FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite Clonase strategy utilizing modified MuLE entry vectors.

    Article Snippet: LR Clonase II Plus (Thermo 12538) reactions were performed with 2 μl Clonase LR II Plus, 20 fmoles pLenti X1 Puro DEST, 10 fmoles of each sgRNA pENTR and 10 fmoles of pENTR hCas9D10A all to 10 μl volume in Tris-EDTA (TE) buffer pH 8.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Modification

    Overview of the lentiviral (A) or retroviral (B) vectors for protein expression or depletion. Entry vectors can be recombined using the LR clonase into the Destination vectors connected by arrows. For RNAi-mediated depletion, miRNA-based and shRNA-based vectors are available for either constitutive or inducible depletions (see Figure 2A for details). For expression, various tags are available (see Figure 2B for details). The RNAi cassettes can be inserted in the middle of the lentiviral backbone (pLenti X1 series), in the 3′ LTR (pLenti X2 series) or in a backbone expressing GFP (pLenti CMV GFP). For RNAi studies using retroviruses, all the Destination cassettes have been inserted in the 3′ LTR of the vectors. For protein expression, the EF-1α, PGK or CMV promoters are available for lentiviruses. CMV-driven constructs can be either constitutive or inducible. For the retroviral vectors, no constitutive expression of cDNAs can be attained in T-REx cell lines using the pQCXI series vectors because they all contain the inducible CMV/TO promoter. Similarly, the drug resistance gene will be repressed in a T-REx cell line since it is after an IRES element under the control of the CMV/TO promoter. However, the pQCXP CMV/TO can be used for inducible expression of cDNAs under constitutive puromycin selection in T-REx cell lines. See Table 2 for details.

    Journal: PLoS ONE

    Article Title: A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

    doi: 10.1371/journal.pone.0006529

    Figure Lengend Snippet: Overview of the lentiviral (A) or retroviral (B) vectors for protein expression or depletion. Entry vectors can be recombined using the LR clonase into the Destination vectors connected by arrows. For RNAi-mediated depletion, miRNA-based and shRNA-based vectors are available for either constitutive or inducible depletions (see Figure 2A for details). For expression, various tags are available (see Figure 2B for details). The RNAi cassettes can be inserted in the middle of the lentiviral backbone (pLenti X1 series), in the 3′ LTR (pLenti X2 series) or in a backbone expressing GFP (pLenti CMV GFP). For RNAi studies using retroviruses, all the Destination cassettes have been inserted in the 3′ LTR of the vectors. For protein expression, the EF-1α, PGK or CMV promoters are available for lentiviruses. CMV-driven constructs can be either constitutive or inducible. For the retroviral vectors, no constitutive expression of cDNAs can be attained in T-REx cell lines using the pQCXI series vectors because they all contain the inducible CMV/TO promoter. Similarly, the drug resistance gene will be repressed in a T-REx cell line since it is after an IRES element under the control of the CMV/TO promoter. However, the pQCXP CMV/TO can be used for inducible expression of cDNAs under constitutive puromycin selection in T-REx cell lines. See Table 2 for details.

    Article Snippet: LR recombination and purification of the plasmid DNA The LR recombination was performed using the LR clonase mix (cat. #11791-019, Invitrogen) with 1 µl of miniprep DNA for each of the Entry and Destination vector, 4 µl of TE pH 8.0, 2 µl of LR buffer and 2 µl of LR clonase.

    Techniques: Expressing, shRNA, Construct, Selection

    Overview of the viral system. A cDNA/shRNA/miRNA is cloned into an Entry vector between the attL1 and attL2 sites. In the presence of the LR clonase, recombination occurs between attL1-attR1 and attL2-attR2 to transfer the insert from the Entry vector into the Destination vector of choice. All Entry vectors contain the kanamycin resistance gene whereas all Destination vectors carry the ampicillin resistance gene.

    Journal: PLoS ONE

    Article Title: A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

    doi: 10.1371/journal.pone.0006529

    Figure Lengend Snippet: Overview of the viral system. A cDNA/shRNA/miRNA is cloned into an Entry vector between the attL1 and attL2 sites. In the presence of the LR clonase, recombination occurs between attL1-attR1 and attL2-attR2 to transfer the insert from the Entry vector into the Destination vector of choice. All Entry vectors contain the kanamycin resistance gene whereas all Destination vectors carry the ampicillin resistance gene.

    Article Snippet: LR recombination and purification of the plasmid DNA The LR recombination was performed using the LR clonase mix (cat. #11791-019, Invitrogen) with 1 µl of miniprep DNA for each of the Entry and Destination vector, 4 µl of TE pH 8.0, 2 µl of LR buffer and 2 µl of LR clonase.

    Techniques: shRNA, Clone Assay, Plasmid Preparation

    FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite Clonase strategy utilizing modified MuLE entry vectors.

    Journal: Gene

    Article Title: A Splice Junction-Targeted CRISPR Approach (spJCRISPR) Reveals Human FOXO3B To Be A Protein-Coding Gene

    doi: 10.1016/j.gene.2018.06.048

    Figure Lengend Snippet: FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite Clonase strategy utilizing modified MuLE entry vectors.

    Article Snippet: LR Clonase II (Thermo 11791100) reactions were performed with a modified form of the pInducer20 vector (Addgene #44012) where the Neo resistance cassette was replaced with a blasticidin resistance cassette.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Modification

    Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP Clonase reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.

    Journal: PLoS ONE

    Article Title: Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System

    doi: 10.1371/journal.pone.0145833

    Figure Lengend Snippet: Construction of a recombination cloning plasmid for the production of avidin fusion proteins. (A) Gene for mAvidin was synthesized with restriction sites NdeI and NotI and subcloned into pDEST17 between the 6x histidine and the gateway cassette to generate p17-Avi plasmid. OVA peptide 323-339 and ESAT6 DNA sequences terminated with att B sites were cloned into pDONR221 via BP Clonase reaction. Thereafter, genes of interest were subcloned into p17-Avi by mean of one step LR Clonase reaction. (B) E . coli BL21 was transformed with p17-Avi encoding ESAT6 and OVA peptide 323-339 . Recombinant Avi-proteins were purified from inclusion bodies and subjected to 12% SDS-PAGE gel and EZ blue staining to analyze the quality of fusion protein preparations. Expected sizes for Avi-ESAT6 and Avi-OVA 757-1037 are 26.8 and 27.3 kDa respectively.

    Article Snippet: Thereafter, OVA DNA ORF was transferred into p17-Avi through site-specific in vitro recombination using the LR clonase (Invitrogen).

    Techniques: Clone Assay, Plasmid Preparation, Avidin-Biotin Assay, Synthesized, Transformation Assay, Recombinant, Purification, SDS Page, Staining

    Construction of tagged transposon mutants in C. albicans . (A) A commercial Tn5 transposon was modified by inclusion of a Gateway conversion cassette (with the ccdB selection gene and chloramphenicol resistance gene (Cm R )), a kanamycin resistance gene (Kan R ), and the UAU1 marker cassette at the multiple cloning site (MCS). A reaction with the modified Tn5, a pool of TagModules, and LR clonase induces recombination at the att sites, placing the TagModule within the transposon mosaic ends (Tn5L and Tn5R). (B) To generate the heterozygous disruption strains, multiple genomic DNA libraries were constructed and then mutagenized in vitro with the pool of tagged Tn5 transposons from (A). Plasmids containing individual insertions were recovered from E. coli and sequenced (arrow) to determine the disrupted gene and its corresponding tag. Results were sorted to maximize the unique gene insertions/tag pairs. Select insertions were amplified, followed by excision of the genomic DNA containing the tagged transposon insertion. Transformation of these inserts into C. albicans was mediated by homology of the genomic sequence flanking the transposon insertion, and integration was selected for based on the arginine prototrophy conferred by the Arg+ marker. Homologous recombination results in a gene disruption with a tagged Tn5 transposon. (C) Screening methodology. Approximately equivalent numbers of tagged transposon mutants are combined to create a tagged pool. Pools are then screened in diverse conditions, generally over 20 generations of competitive growth with pools being harvested and diluted every 5 generations (5 g, 10 g, 15 g, 20 g). The genomic DNA is then extracted from the pool and the uptags and downtags amplified separately using the common priming sites. After the pooled growth assay, the amplicons are then hybridized to an Affymetrix TAG4 microarray to calculate tag intensity as a proxy for strain abundance.

    Journal: PLoS Pathogens

    Article Title: Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection

    doi: 10.1371/journal.ppat.1001140

    Figure Lengend Snippet: Construction of tagged transposon mutants in C. albicans . (A) A commercial Tn5 transposon was modified by inclusion of a Gateway conversion cassette (with the ccdB selection gene and chloramphenicol resistance gene (Cm R )), a kanamycin resistance gene (Kan R ), and the UAU1 marker cassette at the multiple cloning site (MCS). A reaction with the modified Tn5, a pool of TagModules, and LR clonase induces recombination at the att sites, placing the TagModule within the transposon mosaic ends (Tn5L and Tn5R). (B) To generate the heterozygous disruption strains, multiple genomic DNA libraries were constructed and then mutagenized in vitro with the pool of tagged Tn5 transposons from (A). Plasmids containing individual insertions were recovered from E. coli and sequenced (arrow) to determine the disrupted gene and its corresponding tag. Results were sorted to maximize the unique gene insertions/tag pairs. Select insertions were amplified, followed by excision of the genomic DNA containing the tagged transposon insertion. Transformation of these inserts into C. albicans was mediated by homology of the genomic sequence flanking the transposon insertion, and integration was selected for based on the arginine prototrophy conferred by the Arg+ marker. Homologous recombination results in a gene disruption with a tagged Tn5 transposon. (C) Screening methodology. Approximately equivalent numbers of tagged transposon mutants are combined to create a tagged pool. Pools are then screened in diverse conditions, generally over 20 generations of competitive growth with pools being harvested and diluted every 5 generations (5 g, 10 g, 15 g, 20 g). The genomic DNA is then extracted from the pool and the uptags and downtags amplified separately using the common priming sites. After the pooled growth assay, the amplicons are then hybridized to an Affymetrix TAG4 microarray to calculate tag intensity as a proxy for strain abundance.

    Article Snippet: Correct clones were then transferred to the pAG416GPD-ccdB destination vector using the LR clonase reaction (Invitrogen), and resequenced as described.

    Techniques: Modification, Selection, Marker, Clone Assay, Construct, In Vitro, Amplification, Transformation Assay, Sequencing, Homologous Recombination, Growth Assay, Microarray