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  • 93
    Lonza dna ladder 50 bp 1 5 kb
    Dna Ladder 50 Bp 1 5 Kb, supplied by Lonza, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs hot start phusion polymerase
    Hot Start Phusion Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Qiagen qx alignment marker 50 bp 5kb
    Qx Alignment Marker 50 Bp 5kb, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher kb plus dna ladder
    Kb Plus Dna Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1967 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc meta paired sequencing data
    Meta Paired Sequencing Data, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti mouse mhci h2 kb pe
    Anti Mouse Mhci H2 Kb Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore onoo
    Onoo, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology pnf kb p65
    Pnf Kb P65, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr buffer
    Pcr Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 7741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    af6  (ATCC)
    88
    ATCC af6
    Af6, supplied by ATCC, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore pzdonor
    AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and <t>pZDonor</t> with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.
    Pzdonor, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher biotin 14 dctp
    AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and <t>pZDonor</t> with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.
    Biotin 14 Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    CLC Bio clc genomics workbench version 5 0 software
    AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and <t>pZDonor</t> with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.
    Clc Genomics Workbench Version 5 0 Software, supplied by CLC Bio, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Graph Pad Software Inc prism 5
    AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and <t>pZDonor</t> with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.
    Prism 5, supplied by Graph Pad Software Inc, used in various techniques. Bioz Stars score: 99/100, based on 6923 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore hybridization probe
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Hybridization Probe, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene pbluescript
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Pbluescript, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 7808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Qiagen qx alignment marker 50
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Qx Alignment Marker 50, supplied by Qiagen, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc 5 0 kb median interprobe spacing
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    5 0 Kb Median Interprobe Spacing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher solid mate paired library construction kit
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Solid Mate Paired Library Construction Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Agilent technologies agilent 2100 bionalyzer
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Agilent 2100 Bionalyzer, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pegfp n1
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Pegfp N1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 160058 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 5 0 kb noti bamhi fragment
    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, <t>DIG-labeled</t> marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) <t>RT-PCR</t> confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.
    5 0 Kb Noti Bamhi Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and pZDonor with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.

    Journal: Molecular Therapy

    Article Title: Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

    doi: 10.1038/mt.2015.223

    Figure Lengend Snippet: AAVS1 locus-specific integration of different size donor DNAs . ( a ) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and pZDonor with or without AAVS1 ZFN mRNA. (Left): Brightfield and fluorescence images of transfected cells. Bar = 100 µm (Right): PCR spanning the integration junction (top) and RFLP assay (bottom) performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVS1 locus. ( b ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZdonor EGFP. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. ( c ) Accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pZDonor Hybrid FVIII. Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in blue; Enhanced Sharkey AAVS1 ZFN recognition half-sites are underlined in red. Control PCR amplified a 900-bp region of the AAVS1 locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity.

    Article Snippet: Three plasmids having a neomycin resistance marker were used to integrate donor DNAs of increasing sizes into intron 1 of PPP1R12C ( Supplementary Figure S1b ): pZDonor (contains 1.5-kb homology to the AAVS1 locus bisected by a 50-bp multiple cloning site; Sigma-Aldrich) pZDonor EGFP (3.75-kb CMV-promoter-GFP excised from pEGFP-C1 (Clontech, Mountain View, CA) cloned in pZDonor) pZDonor Hybrid FVIII (9.1-kb donor encoding human ferritin light chain promoter-hybrid FVIII cDNA cloned in pZDonor; described below).

    Techniques: Transfection, Fluorescence, Polymerase Chain Reaction, RFLP Assay, Amplification, Selection, Sequencing, Plasmid Preparation

    Comparison of site-specific cleavage activities of ZFN constructs . ( a ) Comparison of AAVS1 ZFN variants and transient hypothermia on cleavage efficiency. Genomic DNA from K562 cells which were coelectroporated with pZDonor and the following AAVS1 ZFN variants: obligate heterodimer (OH), Sharkey or Enhanced Sharkey (see Supplementary Figure S1 legend for details) and cultured at either 37 °C or 30 °C. pEGFP in each electroporation served as an index of transfection efficiency. Site-specific cleavage was evaluated by restriction fragment length polymorphism. Results shown are the mean ± SD of triplicate densitometric measurements of the AAVS1 modified locus expressed as a percentage of the combined unmodified and modified locus. ( b ) Graphical representation of panel a data. Different percentages of K562 genomic DNA attaining AAVS1-specific integration of the 50-bp donor DNA for each ZFN variant (determined from panel a ) (left axis) are shown against comparable efficiencies of pEGFP gene transfer by electroporation (right axis). Data are mean ± SD ( n = 3). Integration was significantly greater ( P

    Journal: Molecular Therapy

    Article Title: Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

    doi: 10.1038/mt.2015.223

    Figure Lengend Snippet: Comparison of site-specific cleavage activities of ZFN constructs . ( a ) Comparison of AAVS1 ZFN variants and transient hypothermia on cleavage efficiency. Genomic DNA from K562 cells which were coelectroporated with pZDonor and the following AAVS1 ZFN variants: obligate heterodimer (OH), Sharkey or Enhanced Sharkey (see Supplementary Figure S1 legend for details) and cultured at either 37 °C or 30 °C. pEGFP in each electroporation served as an index of transfection efficiency. Site-specific cleavage was evaluated by restriction fragment length polymorphism. Results shown are the mean ± SD of triplicate densitometric measurements of the AAVS1 modified locus expressed as a percentage of the combined unmodified and modified locus. ( b ) Graphical representation of panel a data. Different percentages of K562 genomic DNA attaining AAVS1-specific integration of the 50-bp donor DNA for each ZFN variant (determined from panel a ) (left axis) are shown against comparable efficiencies of pEGFP gene transfer by electroporation (right axis). Data are mean ± SD ( n = 3). Integration was significantly greater ( P

    Article Snippet: Three plasmids having a neomycin resistance marker were used to integrate donor DNAs of increasing sizes into intron 1 of PPP1R12C ( Supplementary Figure S1b ): pZDonor (contains 1.5-kb homology to the AAVS1 locus bisected by a 50-bp multiple cloning site; Sigma-Aldrich) pZDonor EGFP (3.75-kb CMV-promoter-GFP excised from pEGFP-C1 (Clontech, Mountain View, CA) cloned in pZDonor) pZDonor Hybrid FVIII (9.1-kb donor encoding human ferritin light chain promoter-hybrid FVIII cDNA cloned in pZDonor; described below).

    Techniques: Construct, Cell Culture, Electroporation, Transfection, Modification, Variant Assay

    Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, DIG-labeled marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) RT-PCR confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.

    Journal: Frontiers in Plant Science

    Article Title: RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA

    doi: 10.3389/fpls.2019.00313

    Figure Lengend Snippet: Characterization of transplastomic tobacco plants. (A) Southern blot confirming homoplastomy of the transformed tobacco lines. Tobacco DNA was cut with restriction enzymes and separated on an agarose gel for blotting. A probe hybridizing to homologous recombination regions in tobacco plastids was used to confirm insertion/integration of dsRNA expression cassettes into plastid DNA. A single band of the expected size was observed in each DNA sample; 1, DIG-labeled marker; 2 DNA from WT tobacco; 3, DNA from tobacco with v-ATPaseA dsRNA expression cassette; 4, DNA from tobacco with GFP dsRNA expression cassette. All DNA samples display single bands which confirm homoplastomy of transformed plants. (B) RT-PCR confirming expression of v-ATPaseA dsRNA and GFP dsRNA in transplastomic plants. Primers annealing to dsRNA sequences were used to confirm production of dsRNA in transplastomic tobacco plants. Tobacco EF1-α gene was used as a positive control and NRT reactions confirmed absence of DNA contamination in cDNA preparations.

    Article Snippet: The hybridization probe was prepared using the PCR DIG Probe Synthesis Kit (Sigma-Aldrich) with primers ( ) that generated a 1181 bp DIG-labeled probe from the homologous recombination regions.

    Techniques: Southern Blot, Transformation Assay, Agarose Gel Electrophoresis, Homologous Recombination, Expressing, Labeling, Marker, Reverse Transcription Polymerase Chain Reaction, Positive Control