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  • 99
    Thermo Fisher takara dna polymerase
    Takara Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa klenow fragment
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 598 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa e coli dna polymerase i
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    E Coli Dna Polymerase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa klenow fragments
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Klenow Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa t4 dna polymerase i
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    T4 Dna Polymerase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa nde i
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Nde I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa xho i
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Xho I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa pyex bx
    Western blot analysis of yeast extracts. Strains W303 <t>pYEX-BX</t> (empty vector), and W303 pYEX-BX 2.1vhs (2.1vhs) were grown to an OD 600 of 2 to 3 in YNBG and then induced with 0.15 mM CuSO 4 for 5 h at 30°C. Whole-cell extracts were prepared, and the <t>vhs</t> protein in the extracts was detected by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at an MOI of 10 was included as a positive control.
    Pyex Bx, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa pegfp n2
    Western blot analysis of yeast extracts. Strains W303 <t>pYEX-BX</t> (empty vector), and W303 pYEX-BX 2.1vhs (2.1vhs) were grown to an OD 600 of 2 to 3 in YNBG and then induced with 0.15 mM CuSO 4 for 5 h at 30°C. Whole-cell extracts were prepared, and the <t>vhs</t> protein in the extracts was detected by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at an MOI of 10 was included as a positive control.
    Pegfp N2, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa pdsred1 n1
    Western blot analysis of yeast extracts. Strains W303 <t>pYEX-BX</t> (empty vector), and W303 pYEX-BX 2.1vhs (2.1vhs) were grown to an OD 600 of 2 to 3 in YNBG and then induced with 0.15 mM CuSO 4 for 5 h at 30°C. Whole-cell extracts were prepared, and the <t>vhs</t> protein in the extracts was detected by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at an MOI of 10 was included as a positive control.
    Pdsred1 N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa pegfp c1
    Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including <t>pEGFP-TAF4</t> (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or <t>pEGFP-C1</t> (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
    Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 880 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa rtaq polymerase
    Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including <t>pEGFP-TAF4</t> (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or <t>pEGFP-C1</t> (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
    Rtaq Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (TaKaRa)
    99
    TaKaRa gfp
    Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including <t>pEGFP-TAF4</t> (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or <t>pEGFP-C1</t> (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
    Gfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 9234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Article Snippet: A fragment containing MxA gene was prepared from pET14b-MxA by digestion with NdeI followed by treatment with Klenow fragment and digestion with BamHI.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

    doi: 10.1016/j.matbio.2016.04.002

    Figure Lengend Snippet: Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Article Snippet: To identify the mutation in the RCS-Cas9 clone #7, the same PCR product was also amplified but with a different DNA polymerase, TaKaRa LA Taq® DNA polymerase (Clontech), and this PCR product cloned into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Labeling, Clone Assay, Genomic Sequencing

    Comparison between DNA preparations and between DNA polymerases DNA preparation methods were as follows: (A) DNA extraction by a simple “boiling” method, (B) DNA extraction by a “rinsing and boiling” method, (C) DNA extraction by a “protein fixation-1, rinsing and boiling” method, (D) DNA extraction by a “protein fixation-2, rinsing and boiling” method, and (E) No DNA extraction process. (A), (B), (C) and (D) were grouped into the “DNA extraction” method, while (E) was grouped into the “non-DNA-extraction” method. DNA polymerases tested in this study were TaKaRa Ex Taq ™ and KOD FX Neo ™ .

    Journal: Kobe Journal of Medical Sciences

    Article Title: Newborn Screening for Spinal Muscular Atrophy: DNA Preparation from Dried Blood Spot and DNA Polymerase Selection in PCR

    doi:

    Figure Lengend Snippet: Comparison between DNA preparations and between DNA polymerases DNA preparation methods were as follows: (A) DNA extraction by a simple “boiling” method, (B) DNA extraction by a “rinsing and boiling” method, (C) DNA extraction by a “protein fixation-1, rinsing and boiling” method, (D) DNA extraction by a “protein fixation-2, rinsing and boiling” method, and (E) No DNA extraction process. (A), (B), (C) and (D) were grouped into the “DNA extraction” method, while (E) was grouped into the “non-DNA-extraction” method. DNA polymerases tested in this study were TaKaRa Ex Taq ™ and KOD FX Neo ™ .

    Article Snippet: As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™ .

    Techniques: DNA Extraction

    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Article Snippet: PCR was performed using DNA polymerase from T.aquaticus (Taq ) or T.thermophilus (Tth ) according to the manufacturer's instructions [rTaq or ExTaq DNA polymerase plus ‘hot start’ antibody (Takara-bio)].

    Techniques: Polymerase Chain Reaction, Genomic Sequencing, Sequencing, Staining

    Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Article Snippet: PCR was performed using DNA polymerase from T.aquaticus (Taq ) or T.thermophilus (Tth ) according to the manufacturer's instructions [rTaq or ExTaq DNA polymerase plus ‘hot start’ antibody (Takara-bio)].

    Techniques: Polymerase Chain Reaction, Staining, Molecular Weight

    Northern blot analysis of CPE-R. Total RNA samples (15 μg/lane) from Vero (lane 1 ), K562 (lane 2 ), Hep3B (lane 3 ), JY (lane 4 ), and Intestine 407 (lane 5 ) cell lines were subjected to Northern blot analysis. The blots were hybridized with 32 Plabeled DNA probes from the entire sequence of clone 706 containing CPE-R cDNA ( top ). The blots were rehybridized with 32 P-labeled human EF-1α probe ( bottom ) to confirm the amounts and integrities of the samples. The positions of 28 and 18S ribosomal RNA are indicated on the right.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Cloning and Functional Characterization of the Receptor for Clostridium perfringens Enterotoxin

    doi:

    Figure Lengend Snippet: Northern blot analysis of CPE-R. Total RNA samples (15 μg/lane) from Vero (lane 1 ), K562 (lane 2 ), Hep3B (lane 3 ), JY (lane 4 ), and Intestine 407 (lane 5 ) cell lines were subjected to Northern blot analysis. The blots were hybridized with 32 Plabeled DNA probes from the entire sequence of clone 706 containing CPE-R cDNA ( top ). The blots were rehybridized with 32 P-labeled human EF-1α probe ( bottom ) to confirm the amounts and integrities of the samples. The positions of 28 and 18S ribosomal RNA are indicated on the right.

    Article Snippet: PolyA+ RNA was reverse transcribed by Superscript reverse transcriptase II ( GIBCO BRL ) at 45°C and then converted to double-stranded cDNA with the aid of RNase H, E. coli DNA polymerase, and E. coli DNA ligase (Takara Shuzo Co., Shiga, Japan).

    Techniques: Northern Blot, Sequencing, Labeling

    Polymerase chain reaction (PCR) template DNA and dsRNA. (A) Diagram illustrating the scheme for producing T7 promoter-flanked templates by PCR. In panel 1 , primers, designed with T7 promoter sequences at their 5′ ends, are used to amplify a DNA fragment from genomic or cDNA sources. Panel 2 shows the PCR product, with the T7 promoter flanks, to be used for transcription of dsRNA by T7 RNA polymerase. Panel 3 illustrates the dsRNA product that is produced from the transcription of both strands of the template drawn in panel 2 . (B) PCR amplification of a 938-bp segment of cnn cDNA with T7 flanking sequences ( lane 1 ). Transcription from the PCR fragment with T7 polymerase yields a double-stranded RNA product that migrates slower on a gel than the double-stranded DNA template ( lane 2 ). The sample in lane 2 was treated with DNase I prior to loading. dsRNA samples typically produce a smear on an agarose gel like that shown in lane 2 . DNA size markers (1-kb ladder) are shown in lane 3 .

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: RNAi in Cultured Drosophila Cells

    doi:

    Figure Lengend Snippet: Polymerase chain reaction (PCR) template DNA and dsRNA. (A) Diagram illustrating the scheme for producing T7 promoter-flanked templates by PCR. In panel 1 , primers, designed with T7 promoter sequences at their 5′ ends, are used to amplify a DNA fragment from genomic or cDNA sources. Panel 2 shows the PCR product, with the T7 promoter flanks, to be used for transcription of dsRNA by T7 RNA polymerase. Panel 3 illustrates the dsRNA product that is produced from the transcription of both strands of the template drawn in panel 2 . (B) PCR amplification of a 938-bp segment of cnn cDNA with T7 flanking sequences ( lane 1 ). Transcription from the PCR fragment with T7 polymerase yields a double-stranded RNA product that migrates slower on a gel than the double-stranded DNA template ( lane 2 ). The sample in lane 2 was treated with DNase I prior to loading. dsRNA samples typically produce a smear on an agarose gel like that shown in lane 2 . DNA size markers (1-kb ladder) are shown in lane 3 .

    Article Snippet: Thermostable DNA polymerase (e.g., Clontech Advantage2 PCR reagent).

    Techniques: Polymerase Chain Reaction, Produced, Amplification, Agarose Gel Electrophoresis

    Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with ( A ) Taq DNA polymerase, ( B ) Tth DNA polymerase, ( C ) Vent(exo-) DNA polymerase, ( D ) KOD Dash DNA polymerase and ( E ) KOD(exo-) DNA polymerase. The x -axis indicates the kind of triphosphate analog used, and the y -axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.

    Journal: Nucleic Acids Research

    Article Title: Systematic characterization of 2?-deoxynucleoside- 5?-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

    doi: 10.1093/nar/gkl637

    Figure Lengend Snippet: Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with ( A ) Taq DNA polymerase, ( B ) Tth DNA polymerase, ( C ) Vent(exo-) DNA polymerase, ( D ) KOD Dash DNA polymerase and ( E ) KOD(exo-) DNA polymerase. The x -axis indicates the kind of triphosphate analog used, and the y -axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.

    Article Snippet: Materials The following commercial available thermostable DNA polymerases were purchased: Taq (Takara Bio), Tth (Toyobo), Vent(exo-) (New England Biolabs) and KOD Dash (Toyobo).

    Techniques: Modification, Generated, Polymerase Chain Reaction

    Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from pUC18 (amplifying region I) and the C5-modified nucleoside triphosphates. ( A–C ) The reaction mixtures containing dATP, dGTP, dCTP and T AL (lane 3), T AC (lane 4), T AF (lane 5), T PA (lane 6), T PN (lane 7), T PR (lane 8), T A2 (lane 11), T A4 (lane 12), T A6 (lane 13), T G6 (lane 14), T ME (lane 15), T CN (lane 16), or T DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). ( D–F ) Reaction mixtures containing dATP, dGTP, TTP and C AL (lane 3), C AC (lane 4), C AF (lane 5), C PA (lane 6), C PN (lane 7), C PR (lane 8), C A2 (lane 11), C A4 (lane 12), C A6 (lane 13), C G6 (lane 14), C ME (lane 15), C CN (lane 16) or C DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable DNA polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).

    Journal: Nucleic Acids Research

    Article Title: Systematic characterization of 2?-deoxynucleoside- 5?-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

    doi: 10.1093/nar/gkl637

    Figure Lengend Snippet: Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from pUC18 (amplifying region I) and the C5-modified nucleoside triphosphates. ( A–C ) The reaction mixtures containing dATP, dGTP, dCTP and T AL (lane 3), T AC (lane 4), T AF (lane 5), T PA (lane 6), T PN (lane 7), T PR (lane 8), T A2 (lane 11), T A4 (lane 12), T A6 (lane 13), T G6 (lane 14), T ME (lane 15), T CN (lane 16), or T DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). ( D–F ) Reaction mixtures containing dATP, dGTP, TTP and C AL (lane 3), C AC (lane 4), C AF (lane 5), C PA (lane 6), C PN (lane 7), C PR (lane 8), C A2 (lane 11), C A4 (lane 12), C A6 (lane 13), C G6 (lane 14), C ME (lane 15), C CN (lane 16) or C DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable DNA polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).

    Article Snippet: Materials The following commercial available thermostable DNA polymerases were purchased: Taq (Takara Bio), Tth (Toyobo), Vent(exo-) (New England Biolabs) and KOD Dash (Toyobo).

    Techniques: Staining, Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Modification, Electrophoresis, Positive Control, Generated, Negative Control

    Optimization of iTaq (DNA polymerase), dNTPs, and Mg 2+ for the duplex real-time PCR. (A) Amplification data of the duplex real-time PCR system directly combined by two single-plex systems without any extra reagents. RFU means relative fluorescence units. (B) Amplification data of V 2 – 5 phage at different concentrations of additional MgCl 2 and dNTPs and (C) DNA polymerase (iTaq).

    Journal: Frontiers in Microbiology

    Article Title: Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration

    doi: 10.3389/fmicb.2019.03023

    Figure Lengend Snippet: Optimization of iTaq (DNA polymerase), dNTPs, and Mg 2+ for the duplex real-time PCR. (A) Amplification data of the duplex real-time PCR system directly combined by two single-plex systems without any extra reagents. RFU means relative fluorescence units. (B) Amplification data of V 2 – 5 phage at different concentrations of additional MgCl 2 and dNTPs and (C) DNA polymerase (iTaq).

    Article Snippet: DNA polymerase (iTaq), Mg2+ , dNTPs, 6× loading buffer, and DNA marker were bought from Takara Bio (Beijing, China).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Fluorescence

    Schematic representation of the GREM technique (for details, see text). The procedure includes three major stages: (Stage 1) genome-wide amplification of the genomic DNA flanking the 3′ ends of target repetitive elements (here, HS LTRs). Treatment of the resulting amplicon with ExoIII generates 5′ protruding ends to be used at the third stage. (Stage 2) A double-stranded oligo d(T)-primed cDNA library is synthesized for tissues where expression of repetitive elements is to be studied. At this stage cDNAs are tagged by a linker oligonucleotide (CS) at the RNA transcription start sites using the ‘cap-switch’ effect. cDNAs are then digested with AluI restriction endonuclease that has no recognition sites within HS LTRs. This step precludes amplification of LTR sequences read-through in the sense orientation. (Stage 3) Finally, the genomic DNA amplicon (Stage 1) is hybridized to the 5′ tagged cDNAs (Stage 2). The protruding DNA ends are filled in with DNA polymerase, and the hybrids obtained (ELTs) are nested PCR amplified with primers specific to the flanking genomic DNA adapter and cDNA 5′ terminal tag sequence, respectively.

    Journal: Nucleic Acids Research

    Article Title: GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats

    doi: 10.1093/nar/gkl335

    Figure Lengend Snippet: Schematic representation of the GREM technique (for details, see text). The procedure includes three major stages: (Stage 1) genome-wide amplification of the genomic DNA flanking the 3′ ends of target repetitive elements (here, HS LTRs). Treatment of the resulting amplicon with ExoIII generates 5′ protruding ends to be used at the third stage. (Stage 2) A double-stranded oligo d(T)-primed cDNA library is synthesized for tissues where expression of repetitive elements is to be studied. At this stage cDNAs are tagged by a linker oligonucleotide (CS) at the RNA transcription start sites using the ‘cap-switch’ effect. cDNAs are then digested with AluI restriction endonuclease that has no recognition sites within HS LTRs. This step precludes amplification of LTR sequences read-through in the sense orientation. (Stage 3) Finally, the genomic DNA amplicon (Stage 1) is hybridized to the 5′ tagged cDNAs (Stage 2). The protruding DNA ends are filled in with DNA polymerase, and the hybrids obtained (ELTs) are nested PCR amplified with primers specific to the flanking genomic DNA adapter and cDNA 5′ terminal tag sequence, respectively.

    Article Snippet: Another possible problem of PCR selection in favor of GC-rich sequences was solved using highly processive DNA polymerases (Clontech Advantage Polymerase Mix).

    Techniques: Genome Wide, Amplification, cDNA Library Assay, Synthesized, Expressing, Nested PCR, Sequencing

    Agarose gel electrophoresis of polymerase chain reaction products stained with ethidium bromide. Lane 1, DNA marker; lanes 2, 3, 4, 5, and 6, amplified hpaA gene using pfu DNA polymerase.

    Journal: Clinical and Experimental Vaccine Research

    Article Title: Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target

    doi: 10.7774/cevr.2016.5.1.19

    Figure Lengend Snippet: Agarose gel electrophoresis of polymerase chain reaction products stained with ethidium bromide. Lane 1, DNA marker; lanes 2, 3, 4, 5, and 6, amplified hpaA gene using pfu DNA polymerase.

    Article Snippet: Polymerase chain reactions (PCRs) were performed in a total volume of 25 µL using 0.5 mM of each primer, 5 mL of PCR buffer (5×), 0.2 mM of each dNTP, 2.5 units of DNA polymerase Pfu enzyme (Takara, Tokyo, Japan) and 200 ng of DNA template.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining, Marker, Amplification

    Western blot analysis of yeast extracts. Strains W303 pYEX-BX (empty vector), and W303 pYEX-BX 2.1vhs (2.1vhs) were grown to an OD 600 of 2 to 3 in YNBG and then induced with 0.15 mM CuSO 4 for 5 h at 30°C. Whole-cell extracts were prepared, and the vhs protein in the extracts was detected by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at an MOI of 10 was included as a positive control.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Western blot analysis of yeast extracts. Strains W303 pYEX-BX (empty vector), and W303 pYEX-BX 2.1vhs (2.1vhs) were grown to an OD 600 of 2 to 3 in YNBG and then induced with 0.15 mM CuSO 4 for 5 h at 30°C. Whole-cell extracts were prepared, and the vhs protein in the extracts was detected by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at an MOI of 10 was included as a positive control.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Western Blot, Plasmid Preparation, Infection, Positive Control

    Levels of expression of 2.1vhs and 2.1vhs1 proteins. Strains W303 pYEX-BX (empty vector), W303 pYEX-BX 2.1vhs (2.1vhs), and W303 pYEX-BX 2.1vhs1 (2.1vhs1) were grown to saturation in YNBG, and the cultures were then split into two portions. One culture was induced with 0.5 mM CuSO 4 for 5 h at 30°C (lanes I), while the other was left untreated (lanes U). Cell extracts were then analyzed for vhs expression by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at a multiplicity of infection (MOI) of 10 was included as a positive control.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Levels of expression of 2.1vhs and 2.1vhs1 proteins. Strains W303 pYEX-BX (empty vector), W303 pYEX-BX 2.1vhs (2.1vhs), and W303 pYEX-BX 2.1vhs1 (2.1vhs1) were grown to saturation in YNBG, and the cultures were then split into two portions. One culture was induced with 0.5 mM CuSO 4 for 5 h at 30°C (lanes I), while the other was left untreated (lanes U). Cell extracts were then analyzed for vhs expression by Western blot analysis using a monoclonal antibody directed against the HA epitope. A lysate of Vero cells infected for 12 h with HSV-1 Pvhs N138-HA (lane Pvhs N138-HA) at a multiplicity of infection (MOI) of 10 was included as a positive control.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Expressing, Plasmid Preparation, Western Blot, Infection, Positive Control

    Expression of vhs from the inducible CUP1 promoter inhibits colony formation. Strains W303 pYEX-BX (vector), W303 pYEX-BX vhs (vhs), and W303 pYEX-BX vhs1 (vhs1) were grown to saturation in YNBD, and then equal amounts of cells from each strain were spotted and streaked onto YNBD (glucose), YNBG (galactose), YNBR (raffinose), and YNBM (maltose) plates containing (+) or lacking (−) 0.5 mM copper sulfate (Cu). The plates were incubated at 30°C for 5 days.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Expression of vhs from the inducible CUP1 promoter inhibits colony formation. Strains W303 pYEX-BX (vector), W303 pYEX-BX vhs (vhs), and W303 pYEX-BX vhs1 (vhs1) were grown to saturation in YNBD, and then equal amounts of cells from each strain were spotted and streaked onto YNBD (glucose), YNBG (galactose), YNBR (raffinose), and YNBM (maltose) plates containing (+) or lacking (−) 0.5 mM copper sulfate (Cu). The plates were incubated at 30°C for 5 days.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Expressing, Plasmid Preparation, Incubation

    Carbon source-dependent variation in the vhs-induced phenotype in liquid cultures. Strains W303 pYEX-BX (vector), W303 pYEX-BX vhs (vhs), and W303 pYEX-BX vhs1 (vhs1) growing in YNBD (glucose) were pelleted, washed in water, and resuspended in YNB containing the indicated carbon sources, in the presence or absence of 0.5 mM CuSO 4 . The OD 600 of the cultures was then monitored over time. Closed symbols, not induced; open symbols, induced. Circles, empty vector; squares, vhs expression vector; triangles, vhs1 expression vector.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Carbon source-dependent variation in the vhs-induced phenotype in liquid cultures. Strains W303 pYEX-BX (vector), W303 pYEX-BX vhs (vhs), and W303 pYEX-BX vhs1 (vhs1) growing in YNBD (glucose) were pelleted, washed in water, and resuspended in YNB containing the indicated carbon sources, in the presence or absence of 0.5 mM CuSO 4 . The OD 600 of the cultures was then monitored over time. Closed symbols, not induced; open symbols, induced. Circles, empty vector; squares, vhs expression vector; triangles, vhs1 expression vector.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Plasmid Preparation, Expressing

    Effect of vhs expression on the in vivo stability of PDA1 mRNA. Strains W303 pYEX-BX (empty vector) and W303 pYEX-BX vhs (vhs) were grown to an OD 600 of 0.6 in YNBD, and a 10-ml aliquot was withdrawn for RNA extraction (solid arrow). The remainder of the culture was induced with the addition of 0.5 mM CuSO 4 for 30 min. Another aliquot was then removed for RNA extraction (open arrow), and transcription was inhibited in the remainder of the culture with 100 μg of phenanthroline per ml. Aliquots were then removed every 30 min over a 3-h time course. Total RNA was analyzed for PDA1 mRNA levels by Northern blot hybridization. Lane 1, RNA extracted just before induction with CuSO 4 ; lane 2, RNA extracted just before addition of phenanthroline; lanes 3, 4, 5, 6, 7, and 8, RNA extracted at 30, 60, 90, 120, 180, and 210 min after addition of phenanthroline, respectively.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Effect of vhs expression on the in vivo stability of PDA1 mRNA. Strains W303 pYEX-BX (empty vector) and W303 pYEX-BX vhs (vhs) were grown to an OD 600 of 0.6 in YNBD, and a 10-ml aliquot was withdrawn for RNA extraction (solid arrow). The remainder of the culture was induced with the addition of 0.5 mM CuSO 4 for 30 min. Another aliquot was then removed for RNA extraction (open arrow), and transcription was inhibited in the remainder of the culture with 100 μg of phenanthroline per ml. Aliquots were then removed every 30 min over a 3-h time course. Total RNA was analyzed for PDA1 mRNA levels by Northern blot hybridization. Lane 1, RNA extracted just before induction with CuSO 4 ; lane 2, RNA extracted just before addition of phenanthroline; lanes 3, 4, 5, 6, 7, and 8, RNA extracted at 30, 60, 90, 120, 180, and 210 min after addition of phenanthroline, respectively.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Expressing, In Vivo, Plasmid Preparation, RNA Extraction, Northern Blot, Hybridization

    Expression of epitope-tagged vhs inhibits colony formation. Strains W303 pYEX-BX (vector), W303 pYEX-BX 2.1vhs (2.1vhs), and W303 pYEX-BX 2.1vhs1 (2.1vhs1) were grown to saturation in YNBD, and then equal amounts of cells from each strain were spotted and streaked onto YNBD (glucose), YNBG (galactose), YNBR (raffinose), and YNBM (maltose) plates containing (+) or lacking (−) 0.5 mM copper sulfate (Cu). The plates were incubated at 30°C for 5 days.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

    doi: 10.1128/JVI.75.3.1172-1185.2001

    Figure Lengend Snippet: Expression of epitope-tagged vhs inhibits colony formation. Strains W303 pYEX-BX (vector), W303 pYEX-BX 2.1vhs (2.1vhs), and W303 pYEX-BX 2.1vhs1 (2.1vhs1) were grown to saturation in YNBD, and then equal amounts of cells from each strain were spotted and streaked onto YNBD (glucose), YNBG (galactose), YNBR (raffinose), and YNBM (maltose) plates containing (+) or lacking (−) 0.5 mM copper sulfate (Cu). The plates were incubated at 30°C for 5 days.

    Article Snippet: The Nco I- Hin dIII fragment of pYGAL vhs bearing the vhs ORF and 3′ flanking sequences was cloned between the Bam HI- Sal I sites of pYEX-BX (after making all four ends flush with the Klenow fragment of DNA polymerase I), generating pYEX-BX vhs. pYEX-BX vhs1 was generated in the same way, using the Nco I- Hin dIII fragment of pYGAL vhs1.

    Techniques: Expressing, Plasmid Preparation, Incubation

    Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

    Journal: PLoS ONE

    Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

    doi: 10.1371/journal.pone.0054075

    Figure Lengend Snippet: Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

    Article Snippet: Plasmid pEGFP-TAF4-NM, which encodes the region in TAF4 from amino acid 1 to 701, was constructed by digesting pEGFP-TAF4 with Cla I and Xho I, eluting the digested fragment and treating with the DNA polymerase I Klenow fragment, and then inserted into pEGFP-C1.

    Techniques: Transfection

    Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

    Journal: PLoS ONE

    Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

    doi: 10.1371/journal.pone.0054075

    Figure Lengend Snippet: Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

    Article Snippet: Plasmid pEGFP-TAF4-NM, which encodes the region in TAF4 from amino acid 1 to 701, was constructed by digesting pEGFP-TAF4 with Cla I and Xho I, eluting the digested fragment and treating with the DNA polymerase I Klenow fragment, and then inserted into pEGFP-C1.

    Techniques: Immunofluorescence, Transfection, Incubation, Laser-Scanning Microscopy, Staining