dna polymerase i  (TaKaRa)

 
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    Name:
    Klenow Fragment
    Description:
    DNA Polymerase I Klenow Fragment Large Fragment is a proteolytic product of E coli DNA Polymerase I which possesses the 5 →3 polymerase and 3 →5 exonuclease activities of intact DNA Polymerase I but lacks the 5 →3 exonuclease activity of full length DNA Polymerase I The lack of 5 →3 exonuclease activity enables the DNA Polymerase I Klenow Fragment to be used in random primer labeling and DNA sequencing experiments in addition to second strand cDNA synthesis The enzyme is supplied in a buffer of 50 mM potassium phosphate pH 6 5 1 mmDTT and 50 glyerol 2140A B or ethylene glycol 2140AK BK
    Catalog Number:
    2140b
    Price:
    None
    Size:
    1 000 Units
    Category:
    DNA Polymerase I Klenow Fragment DNA polymerases Modifying enzymes Cloning
    Buy from Supplier


    Structured Review

    TaKaRa dna polymerase i
    Amplification of the ORFV <t>DNA</t> polymerase gene by PCR. Lane M, DNA marker DL-2000; lane 1, DNA <t>polymerase</t> I PCR product (888 bp); lane 2, DNA polymerase II PCR product (1001 bp); lane 3, polymerase III PCR product (1752 bp)
    DNA Polymerase I Klenow Fragment Large Fragment is a proteolytic product of E coli DNA Polymerase I which possesses the 5 →3 polymerase and 3 →5 exonuclease activities of intact DNA Polymerase I but lacks the 5 →3 exonuclease activity of full length DNA Polymerase I The lack of 5 →3 exonuclease activity enables the DNA Polymerase I Klenow Fragment to be used in random primer labeling and DNA sequencing experiments in addition to second strand cDNA synthesis The enzyme is supplied in a buffer of 50 mM potassium phosphate pH 6 5 1 mmDTT and 50 glyerol 2140A B or ethylene glycol 2140AK BK
    https://www.bioz.com/result/dna polymerase i/product/TaKaRa
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    dna polymerase i - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells"

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells

    Journal: Archives of Virology

    doi: 10.1007/s00705-013-1896-z

    Amplification of the ORFV DNA polymerase gene by PCR. Lane M, DNA marker DL-2000; lane 1, DNA polymerase I PCR product (888 bp); lane 2, DNA polymerase II PCR product (1001 bp); lane 3, polymerase III PCR product (1752 bp)
    Figure Legend Snippet: Amplification of the ORFV DNA polymerase gene by PCR. Lane M, DNA marker DL-2000; lane 1, DNA polymerase I PCR product (888 bp); lane 2, DNA polymerase II PCR product (1001 bp); lane 3, polymerase III PCR product (1752 bp)

    Techniques Used: Amplification, Polymerase Chain Reaction, Marker

    Related Articles

    Clone Assay:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI. ..

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: The PCR-amplified Mx1 fragment was digested with NheI (TOYOBO) and cloned into pCHA digested with NheI. pCHA-Mx1ΔC plasmid expressing Mx1 lacking its C-terminal (between amino acid positions 563 and 631) was spontaneously generated during the construction of pCHA-Mx1. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: .. Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

    Amplification:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: The mouse Pol I promoter region was amplified by PCR with primers, 5′-TAA TACGACTCACTATA-3′ and 5′-GTCGGTACCTATCTCCAGGTCCA-3′ using pMrBKSP11 (a gift from Dr K. Yamamoto) ( ) as a template. .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: The amplified fragment was digested with NcoI and ligated into NcoI-digested pBluescript II vector (Stratagene) containing a Myc tag sequence. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

    Filtration:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa). .. Next, the annealed substrates were purified by Sepharose CL6B gel filtration.

    Synthesized:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: A plasmid vector, from which an artificial influenza virus genome containing luciferase gene of negative polarity as a reporter is synthesized in cells by the mouse DNA-dependent Pol I, was constructed as follows. .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4. .. For real-time reverse transcription-PCR (RT-PCR) analysis, cDNAs were synthesized with SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen).

    Construct:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: A plasmid vector, from which an artificial influenza virus genome containing luciferase gene of negative polarity as a reporter is synthesized in cells by the mouse DNA-dependent Pol I, was constructed as follows. .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: Paragraph title: Plasmid DNA constructs. ... The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS.

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: In order to construct pCAGGS-NP-Myc for expression of Myc epitope-tagged NP, DNA fragments were amplified by PCR with primers, 5′-CCGAATTCCATGGCGTCTCAAGGCACCAAA-3′ and 5′-CGCGCCATGGCATTATCGTATTCCTCTGCA-3′ and a plasmid containing the NP gene as a template. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Electrophoresis:

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: .. Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: After electrophoresis, RNA was transferred to nylon membrane (Schleicher & Schuell), which was cross-linked with UV and hybridized with DNA probes. .. DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4.

    Incubation:

    Article Title: Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp
    Article Snippet: .. The DNA beads were incubated in a 25 μl of Klenow reaction mixture with 4 μM [α-32 P] TTP, 24 μM α-S-dCTP and 2 units of Klenow fragment at 10°C for 30 min. After three washes with BW buffer and one wash with TE, the attached RF64 was completely elongated with 250 μM dNTP and 2 units of Klenow fragment at 10°C for 30 min in a 20 μl Klenow reaction mixture. ..

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Luciferase:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: A plasmid vector, from which an artificial influenza virus genome containing luciferase gene of negative polarity as a reporter is synthesized in cells by the mouse DNA-dependent Pol I, was constructed as follows. .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Activity Assay:

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease
    Article Snippet: .. Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio). ..

    Expressing:

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS. .. The viral proteinase was inactivated by a previously described point mutation (D26N) in the active site in all M-PMV gag-pro-pol expression vectors ( ).

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: In order to construct pCAGGS-NP-Myc for expression of Myc epitope-tagged NP, DNA fragments were amplified by PCR with primers, 5′-CCGAATTCCATGGCGTCTCAAGGCACCAAA-3′ and 5′-CGCGCCATGGCATTATCGTATTCCTCTGCA-3′ and a plasmid containing the NP gene as a template. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Article Title: Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA
    Article Snippet: Paragraph title: Construction of a secretory hybrid-IgG/IgA expression vector ... The pGEM5zf/TCAB2 was digested with Hin dIII and then blunt ends were generated with the Klenow fragment (Takara Bio, Shiga, Japan).

    Modification:

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: A portion of the M-PMV genome , containing the M-PMV gag , pro , and pol open reading frames, was removed from pSHRMX, a modification of the genomic clone, pSHRM15 , by using the Hha I site at nucleotide 486 and an Xho I site engineered into the genome at the end of the pol reading frame where nucleotides 5816 and 5817 (CT) were converted to TC. .. The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS.

    DNA Synthesis:

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease
    Article Snippet: .. Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio). ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: A 69mer (5′-CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCG TAA TCA TGG-3′) was labeled with [γ-32 P]ATP using T4 polynucleotide kinase (TaKaRa), and was purified by Sephadex G-50 chromatography (Probe-Quant; Pharmacia). .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa).

    Countercurrent Chromatography:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: A 69mer (5′-CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCG TAA TCA TGG-3′) was labeled with [γ-32 P]ATP using T4 polynucleotide kinase (TaKaRa), and was purified by Sephadex G-50 chromatography (Probe-Quant; Pharmacia). .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa).

    Transfection:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection. .. The nucleotide sequence of each plasmid was confirmed by DNA sequencing.

    Sequencing:

    Article Title: CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding
    Article Snippet: .. The wild-type probe of the CIDE-A promoter (from −91 to −57) was generated by annealing two complementary oligonucleotides (SpWT: 5′-GCGGGGCTGGCAGGC GGGCGGGGGCGGG GCCGCCG-3′; 5′-GTGCGGCGGC CCCGCCCCCGCCC GCCTGCCAGCCC-3′, the core sequence of the Sp1/Sp3-binding site is underlined) and filling the 3′ recessive ends by repair synthesis with dATP, dTTP, dGTP, [α-32 P]dCTP and the Klenow fragment of DNA polymerase I (Takara). .. The mutated probe of the CIDE-A promoter was generated by the same procedure, except that several oligonucleotides with mutations (SpM1: 5′-GCGGGGCTGGCAGGC GG A C AA GGGCGGG GCCGCCG-3′, 5′-GTGCGGCGGC CCCGCCC TT G T CC GCCTGCCAGCCC-3′; SpM2: 5′-GCGGGGCTGGCAGGC GGGCGGGG A C AA G GCCGCCG-3′, 5′-GTGCGGCGGC C TT G T CCCCGCCC GCCTGCCAGCCC-3′; SpM3: 5′-GCGGGGCTGGCAGGC GG A C AA GG A C AA G GCCGCCG-3′, 5′-GTGCGGCGGC C TT G T CC TT G T CC GCCTGCCAGCCC-3′, mutations are shown in bold) in the core sequence of the Sp1/Sp3-binding site were used.

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: .. The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS. .. The pBKMGXS vector was made with the wild-type M-PMV matrix domain, as well as A18V, R55W, and both A18V and R55W matrix point mutations.

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection. .. The nucleotide sequence of each plasmid was confirmed by DNA sequencing.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. The three sequences were combined to obtain the complete sequence of ORF025.

    Chromatography:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: A 69mer (5′-CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCG TAA TCA TGG-3′) was labeled with [γ-32 P]ATP using T4 polynucleotide kinase (TaKaRa), and was purified by Sephadex G-50 chromatography (Probe-Quant; Pharmacia). .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa).

    Ligation:

    Article Title: Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA
    Article Snippet: The resulting DNA fragments, Jc-TCAB2 and SC-TCAB1 , were ligated with Hin dIII-digested pUC19 (Invitrogen) by means of three-piece ligation, resulting in the production of pUC19/TCAB2 -Jc-SC-TCAB1 . pUC19/TCAB2 -Jc-SC-TCAB1 was digested with Sac II and then ligated with Sac II-digested PCAB , resulting in the production of the pUC19/Jc-SC expression cassette. .. The pGEM5zf/TCAB2 was digested with Hin dIII and then blunt ends were generated with the Klenow fragment (Takara Bio, Shiga, Japan).

    Northern Blot:

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: Paragraph title: Northern blot analysis and real-time RT-PCR. ... DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4.

    Labeling:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa). .. The labeled product was purified by Sephadex G-50 chromatography.

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: .. Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: .. DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4. ..

    Generated:

    Article Title: CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding
    Article Snippet: .. The wild-type probe of the CIDE-A promoter (from −91 to −57) was generated by annealing two complementary oligonucleotides (SpWT: 5′-GCGGGGCTGGCAGGC GGGCGGGGGCGGG GCCGCCG-3′; 5′-GTGCGGCGGC CCCGCCCCCGCCC GCCTGCCAGCCC-3′, the core sequence of the Sp1/Sp3-binding site is underlined) and filling the 3′ recessive ends by repair synthesis with dATP, dTTP, dGTP, [α-32 P]dCTP and the Klenow fragment of DNA polymerase I (Takara). .. The mutated probe of the CIDE-A promoter was generated by the same procedure, except that several oligonucleotides with mutations (SpM1: 5′-GCGGGGCTGGCAGGC GG A C AA GGGCGGG GCCGCCG-3′, 5′-GTGCGGCGGC CCCGCCC TT G T CC GCCTGCCAGCCC-3′; SpM2: 5′-GCGGGGCTGGCAGGC GGGCGGGG A C AA G GCCGCCG-3′, 5′-GTGCGGCGGC C TT G T CCCCGCCC GCCTGCCAGCCC-3′; SpM3: 5′-GCGGGGCTGGCAGGC GG A C AA GG A C AA G GCCGCCG-3′, 5′-GTGCGGCGGC C TT G T CC TT G T CC GCCTGCCAGCCC-3′, mutations are shown in bold) in the core sequence of the Sp1/Sp3-binding site were used.

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: The PCR-amplified Mx1 fragment was digested with NheI (TOYOBO) and cloned into pCHA digested with NheI. pCHA-Mx1ΔC plasmid expressing Mx1 lacking its C-terminal (between amino acid positions 563 and 631) was spontaneously generated during the construction of pCHA-Mx1. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Article Title: Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA
    Article Snippet: .. The pGEM5zf/TCAB2 was digested with Hin dIII and then blunt ends were generated with the Klenow fragment (Takara Bio, Shiga, Japan). ..

    other:

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

    DNA Sequencing:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection. .. The nucleotide sequence of each plasmid was confirmed by DNA sequencing.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: .. Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4. .. For real-time reverse transcription-PCR (RT-PCR) analysis, cDNAs were synthesized with SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen).

    Quantitative RT-PCR:

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: Paragraph title: Northern blot analysis and real-time RT-PCR. ... DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4.

    Recombinant:

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: All recombinant DNA-cloning techniques followed established methods described elsewhere ( ). .. The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS.

    Mutagenesis:

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS. .. The viral proteinase was inactivated by a previously described point mutation (D26N) in the active site in all M-PMV gag-pro-pol expression vectors ( ).

    Isolation:

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: Total RNA was isolated with TRIzol reagent (Invitrogen/Life Technologies) according to the manufacturer's protocol. .. DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4.

    Electrophoretic Mobility Shift Assay:

    Article Title: CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays ... The wild-type probe of the CIDE-A promoter (from −91 to −57) was generated by annealing two complementary oligonucleotides (SpWT: 5′-GCGGGGCTGGCAGGC GGGCGGGGGCGGG GCCGCCG-3′; 5′-GTGCGGCGGC CCCGCCCCCGCCC GCCTGCCAGCCC-3′, the core sequence of the Sp1/Sp3-binding site is underlined) and filling the 3′ recessive ends by repair synthesis with dATP, dTTP, dGTP, [α-32 P]dCTP and the Klenow fragment of DNA polymerase I (Takara).

    Purification:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: A 69mer (5′-CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCG TAA TCA TGG-3′) was labeled with [γ-32 P]ATP using T4 polynucleotide kinase (TaKaRa), and was purified by Sephadex G-50 chromatography (Probe-Quant; Pharmacia). .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa).

    Polymerase Chain Reaction:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: PCR products were phosphorylated with T4 polynucleotide kinase (TOYOBO) and digested with KpnI (TaKaRa). .. This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: In order to construct pCAGGS-NP-Myc for expression of Myc epitope-tagged NP, DNA fragments were amplified by PCR with primers, 5′-CCGAATTCCATGGCGTCTCAAGGCACCAAA-3′ and 5′-CGCGCCATGGCATTATCGTATTCCTCTGCA-3′ and a plasmid containing the NP gene as a template. .. Subsequently, a DNA fragment containing Myc-tagged NP sequence was excised from the pBluescript II and inserted into pCAGGS that had been digested with EcoRI and blunted with Klenow fragment. pSEAP2-control (Clontech) containing a secreted alkaline phosphatase (SEAP) gene under the control of the SV40 early promoter was used as a control for normalization of transfection.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: .. Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

    Article Title: Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA
    Article Snippet: To subclone mouse SC cDNA, PCR was performed using pcDNA3.1/mouse SC as a template, and SC-specific SCF-Xho and SCR-Xba as primers. .. The pGEM5zf/TCAB2 was digested with Hin dIII and then blunt ends were generated with the Klenow fragment (Takara Bio, Shiga, Japan).

    Random Primer DNA Labeling:

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: .. Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Blocking Assay:

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Activated Clotting Time Assay:

    Article Title: Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding
    Article Snippet: A 69mer (5′-CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCG TAA TCA TGG-3′) was labeled with [γ-32 P]ATP using T4 polynucleotide kinase (TaKaRa), and was purified by Sephadex G-50 chromatography (Probe-Quant; Pharmacia). .. Alternatively, a 17mer (5′-GTAAAGACCGACGGCCAGT-3′) was annealed to M13mp18 virion DNA and was labeled with [α-32 P]dCTP and dGTP to produce a 20mer, using Klenow fragment (TaKaRa).

    Plasmid Preparation:

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
    Article Snippet: Paragraph title: Construction of plasmid vectors ... This fragment was cloned into pHH21 containing the promoter region of human ribosomal RNA gene (a gift from Dr Y. Kawaoka) ( , ), which had been digested with BssHII followed by treatment with Klenow fragment (TaKaRa) and subsequent digestion with KpnI.

    Article Title: A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
    Article Snippet: .. The M-PMV sequence was then removed from pSP73GXS with Eco RI sites, blunt ended with Klenow fragment, and ligated with the pBacPAK1 baculovirus transfer vector (Clontech, Palo Alto, Calif.) by using the Bam HI site, blunt ended with Klenow fragment, to create pBKMGXS. .. The pBKMGXS vector was made with the wild-type M-PMV matrix domain, as well as A18V, R55W, and both A18V and R55W matrix point mutations.

    Article Title: In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells
    Article Snippet: .. Cloning and DNA sequencing The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. .. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

    Article Title: Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA
    Article Snippet: Paragraph title: Construction of a secretory hybrid-IgG/IgA expression vector ... The pGEM5zf/TCAB2 was digested with Hin dIII and then blunt ends were generated with the Klenow fragment (Takara Bio, Shiga, Japan).

    Real-time Polymerase Chain Reaction:

    Article Title: Overexpression of Glucose-6-Phosphate Dehydrogenase Is Associated with Lipid Dysregulation and Insulin Resistance in Obesity †
    Article Snippet: DNA probes were labeled by random priming method by using the Klenow fragment of DNA polymerase I (Takara) and [α-32 P]dCTP (Amersham-Pharmacia). cDNAs used as probes were those for G6PD, 6PGD, ME, IDH, ADD1/SREBP1c, FAS, peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, aP2, HSL, and 36B4. .. It was analyzed in a model iCycler Real-Time PCR Detection System (Bio-Rad) with following primer sets: G6PD, sense (5′-CGATGGCAGAGCAGGT-3′) and antisense (5′-GATCTGGTCCTCACG-3′); FAS, sense (5′-TGCTCCCAGCTGCAGGC-3′) and antisense (5′-GCCCGGTAGCTCTGGGTGTA-3′); PPARγ, sense (5′-TTGCTGAACGTGAAGCCCATCGAGG-3′) and antisense (5′-GTCCTTGTAGATCTCCTGGAGCAG-3′); aP2, sense (5′-CAAAATGTGTGATGCCTTTGTG-3′) and antisense (5′-CTCTTCCTTTGGCTCATGCC-3′); TNF-α, sense (5′-GCCACCACGCTCTTCTGCCT-3′) and antisense (5′-CTGATGGTGTGGGTGAGGAG-3′); resistin, sense (5′-CAGAAGGCACAGCAGTCTTG-3′) and antisense (5′-GACCGGAGGACATCAGACAT-3′); adiponectin, sense (5′-GGCAGGAAAGGAGAACCTGG-3′) and antisense (5′-GCCTTGTCCTTCTTGAAGAG-3′); GAPDH, sense (5′-TGCACCACCAACTGCTTAG-3′) and antisense (5′-GGATGCAGGGATGATGTTC-3′); and cyclophilin, sense (5′-CAGACGCCACTGTCGCTTT-3′) and antisense (5′-TGTCTTTGGAACTTTGTCTGCAA-3′).

    Binding Assay:

    Article Title: CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding
    Article Snippet: The wild-type probe of the CIDE-A promoter (from −91 to −57) was generated by annealing two complementary oligonucleotides (SpWT: 5′-GCGGGGCTGGCAGGC GGGCGGGGGCGGG GCCGCCG-3′; 5′-GTGCGGCGGC CCCGCCCCCGCCC GCCTGCCAGCCC-3′, the core sequence of the Sp1/Sp3-binding site is underlined) and filling the 3′ recessive ends by repair synthesis with dATP, dTTP, dGTP, [α-32 P]dCTP and the Klenow fragment of DNA polymerase I (Takara). .. Nuclear extracts containing 10 μg of the protein were preincubated in 20 μl of binding buffer [10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 μg/ml poly(dI-dC)· poly(dI-dC), and 4% glycerol] with or without unlabeled competitor (100-fold molar excess).

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

    Mobility Shift:

    Article Title: Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells
    Article Snippet: .. Electrophoresis mobility shift assay (EMSA) was performed using nuclear extracts from CD4+ T-cells; double-stranded DNA oligomers were labeled with [α-32 P]-dNTP with a Klenow Fragment and Random Primer DNA Labeling Kit v2.0 (Takara Bio, Inc., Shiga, Japan). shows the sequences of the double-stranded DNA oligomers used. .. For blocking of nonspecific DNA-protein binding, 1 μg of nuclear extract was incubated in reaction mixture including 20 mM HEPES-KOH (pH 7.9), 50 mM KCl, 5% glycerol, 1 mM EDTA (pH 8.0), 10 mM DTT, BSA (10 mg/mL), and poly dI-dC (1 mg/mL) for 15 min on ice.

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    TaKaRa fgf 1
    Fig. 7. Co-localization of <t>FGF-1</t> and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.
    Fgf 1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 87/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa xho i s1 treated mouse mtdna
    Linear 11 kb fragments of <t>mtDNA</t> are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with <t>Xho</t> I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).
    Xho I S1 Treated Mouse Mtdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa thermostable dna polymerases
    Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with ( A ) <t>Taq</t> <t>DNA</t> 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.
    Thermostable Dna Polymerases, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Transfection, Staining, Confocal Microscopy

    Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: In Vivo, Transfection, Staining, Quantitation Assay

    Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, Incubation, In Vitro, SDS Page

    Fig. 1. Identification of proteins binding selectively to FGF-1. ( A ) U2OS cells were incubated overnight (16 h) with [ 35 S]cysteine/methionine, then washed and lysed. The cell lysates were rotated for 1 h at 4°C with either MBP (lane 1), MBP–FGF-1 (lane 2) or MBP–IFN-γ (lane 4) covalently bound to Sepharose beads (∼20 µg of protein). The lysates were then transferred to another Eppendorf tube and subjected to a second round of precipitation for 1 h at 4°C with Sepharose-bound MBP–FGF-1. The Sepharose beads were washed, the proteins eluted with SDS sample buffer and the eluates were subjected to SDS–PAGE [12% (w/v) gel] and fluorography. An asterisk marks proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP, and two asterisks mark proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP–IFN-γ. Arrows point to proteins that bind FGF-1 specifically. Also shown are the positions of molecular weight standards. ( B ) The same conditions as in (A), but the rotations with fusion proteins were performed in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of excess free FGF-1 (500 µg). Arrows mark proteins that bound FGF-1 specifically and which were competed out by excess FGF-1.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 1. Identification of proteins binding selectively to FGF-1. ( A ) U2OS cells were incubated overnight (16 h) with [ 35 S]cysteine/methionine, then washed and lysed. The cell lysates were rotated for 1 h at 4°C with either MBP (lane 1), MBP–FGF-1 (lane 2) or MBP–IFN-γ (lane 4) covalently bound to Sepharose beads (∼20 µg of protein). The lysates were then transferred to another Eppendorf tube and subjected to a second round of precipitation for 1 h at 4°C with Sepharose-bound MBP–FGF-1. The Sepharose beads were washed, the proteins eluted with SDS sample buffer and the eluates were subjected to SDS–PAGE [12% (w/v) gel] and fluorography. An asterisk marks proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP, and two asterisks mark proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP–IFN-γ. Arrows point to proteins that bind FGF-1 specifically. Also shown are the positions of molecular weight standards. ( B ) The same conditions as in (A), but the rotations with fusion proteins were performed in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of excess free FGF-1 (500 µg). Arrows mark proteins that bound FGF-1 specifically and which were competed out by excess FGF-1.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, Incubation, SDS Page, Molecular Weight

    Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, Incubation, Lysis, In Vitro, SDS Page

    Fig. 2. ). U2OS cells were lysed and lysate was incubated for 1 h at 4°C with the indicated amount of immobilized MBP–FGF-1 or MBP. The bound proteins were separated by SDS–PAGE, transferred to a PVDF membrane and blotted with anti-CK2β antibodies. The arrow marks the position of CK2β.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 2. ). U2OS cells were lysed and lysate was incubated for 1 h at 4°C with the indicated amount of immobilized MBP–FGF-1 or MBP. The bound proteins were separated by SDS–PAGE, transferred to a PVDF membrane and blotted with anti-CK2β antibodies. The arrow marks the position of CK2β.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Incubation, SDS Page

    Fig. 9. Phosphorylation of FGF-1 and FGF-2 by CK2 and the ability of the growth factors to stimulate autophosphorylation of CK2β. Purified rat liver CK2 was incubated for 20 min at 30°C either alone or with the indicated quantity of FGF-1 (lanes 2–4), FGF-2 (lanes 5–8) or FGF-1(K132E) (lane 9). The samples were then incubated for 2 h with either heparin–Sepharose (lanes 1–7 and 9) or anti-FGF-2 coupled to protein A–Sepharose (lane 8). The bound proteins were eluted with sample buffer, analysed by SDS–PAGE and exposed to a phosphoimager. It should be noted that CK2 binds to heparin–Sepharose independently of FGF.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 9. Phosphorylation of FGF-1 and FGF-2 by CK2 and the ability of the growth factors to stimulate autophosphorylation of CK2β. Purified rat liver CK2 was incubated for 20 min at 30°C either alone or with the indicated quantity of FGF-1 (lanes 2–4), FGF-2 (lanes 5–8) or FGF-1(K132E) (lane 9). The samples were then incubated for 2 h with either heparin–Sepharose (lanes 1–7 and 9) or anti-FGF-2 coupled to protein A–Sepharose (lane 8). The bound proteins were eluted with sample buffer, analysed by SDS–PAGE and exposed to a phosphoimager. It should be noted that CK2 binds to heparin–Sepharose independently of FGF.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Purification, Incubation, SDS Page

    Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, SPR Assay, Injection

    Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, Incubation, SDS Page, In Vitro, Western Blot

    Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.

    Article Snippet: The MBP fusions of FGF-2, FGF-1 and the FGF-1 mutants have been described previously , except for the MBP–FGF-1(S113A) mutant, which was made using PCR-directed mutagenesis with the plasmid MBP-FGF-1 as template. pEGFP-FGF-1 was made by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pEGFP (Clontech). pcDNA3-myc-FGF-1 was constructed by inserting the cDNA for FGF-1 into the multiple cloning site of the vector pcDNA3 (Invitrogen), and the myc tag was added to the N-terminus of FGF-1 by PCR. pcDNA3-myc-FGF-1pts was then constructed by adding the three amino acids SGG and then the type 1 peroxisomal targeting signal (PTS1) SKL to the C-terminus of FGF-1 in pcDNA3-myc-FGF-1 using PCR.

    Techniques: Binding Assay, Construct, Incubation, Radioactivity, Western Blot, Mutagenesis, DNA Synthesis, Activity Assay, SDS Page

    Linear 11 kb fragments of mtDNA are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with Xho I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: Linear 11 kb fragments of mtDNA are released from control mouse liver mtDNA by single-stranded nuclease treatment. The figure shows a schematic diagram of a replication intermediate of mammalian mtDNA, where fork arrest has occurred in the NCR and near O L ; nicking by S1, or other, nuclease can in theory release a linear fragment of mtDNA of ∼11 kb as illustrated. S1 nuclease treatment of Balb C mouse liver mtDNA yielded just such fragments based on a series of probes from around the mouse mitochondrial genome (Panels A – F ). (Panels A–D) represent a single gel: 0.62% agarose, 60 V, 30 h; whereas, the mtDNA of (Panel E) and (Panel F) was separated on a 0.5% agarose gel, at 65 V for 30 h. Treating mouse liver mtDNA with Xho I or Sac I, in addition to S1 nuclease, produced shorter fragments (Panel G , i–iii), after separation at 100 V for 4 h on a 0.8% agarose gel. Probes were assigned lowercase letters (a–f), and the numbers at the foot of the gel panels indicate the span of the probes, based on the revised mouse mtDNA reference sequence ( 17 ).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Agarose Gel Electrophoresis, Produced, Sequencing

    The ends of the prominent linear molecules of mutator mouse mtDNA map to the NCR and in the vicinity of O L . S1 products of mutator mouse mtDNA were recovered from specific regions of agarose gels using Recochips™; repeated separation of some of the material by 1D-AGE (0.8% agarose, 4 h, 100 V) was used to confirm successful recovery (panel A , i and ii; panel D , iii). Fragments were circularized, and the junction amplified, cloned and sequenced. Panel ( B ) A partial linear map of the mouse mitochondrial genome encompassing the NCR. The ends (red diamonds) identified by direct sequencing were nt 16 033, 16 016, 15 997, 15 995, 15 978, 15 956 (Recochip 1); and 15 875, 15 751, 15 751, 15 750, 15 747, 15 529, 15 489, 15 487, 15 486, 15 467 and 15 389 (Recochip 2). Scarcer ends mapping to the NCR were also cloned and sequenced from control littermates (yellow diamonds). The NCR of mouse mtDNA includes three so-called conserved sequenced boxes (CSBs 1–3), a conserved central domain, and a predicted clover-leaf structure, TAS, which is believed to effect termination of 7S DNA (D-loop) synthesis. Three of the ends (15 751, 15 751 and 15 750) in the conserved central domain are flanked by a GC-rich sequence CCGGGCCC and GGGGG, which is extremely rare in the L-strand of mammalian mtDNAs, suggesting that this may represent a cis -element. Free 5′ ends of DNA identified previously by LM-PCR are represented as vertical blue lines; in panel B the free 5′ ends comprise two clusters, cluster I (O H ) and cluster II ( 12 , 13 , 38 ). The height of the most prominent free end (nt 16 034) was set arbitrarily and the others expressed as a fraction of its height. Panel ( C ) The sequenced Sac I/S1 products with an end mapping close to O L (nt 5160–5191) were nt 4946, 5082, 5133, 5201, 5217, 5255, 5257, 5318, 5477 (red diamonds) in mutator mouse samples and nt 5169, 5193, 5196, 5199, 5244 and 5362 (yellow diamonds) in controls. In humans, free 5′ ends are concentrated in the tRNA Cys gene (C), which is adjacent to O L ( 38 , 39 ) and free 5′ ends map to similar positions in mouse mtDNA (see Supplementary Figure 2 ); in panel (C) they are again represented as blue vertical lines. Panel (D) Sac I/S1-treated mutator mouse mtDNA analysed by 1D-AGE, iii is the material that was used to define the ends of DNA represented as red diamonds in panel (C).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: The ends of the prominent linear molecules of mutator mouse mtDNA map to the NCR and in the vicinity of O L . S1 products of mutator mouse mtDNA were recovered from specific regions of agarose gels using Recochips™; repeated separation of some of the material by 1D-AGE (0.8% agarose, 4 h, 100 V) was used to confirm successful recovery (panel A , i and ii; panel D , iii). Fragments were circularized, and the junction amplified, cloned and sequenced. Panel ( B ) A partial linear map of the mouse mitochondrial genome encompassing the NCR. The ends (red diamonds) identified by direct sequencing were nt 16 033, 16 016, 15 997, 15 995, 15 978, 15 956 (Recochip 1); and 15 875, 15 751, 15 751, 15 750, 15 747, 15 529, 15 489, 15 487, 15 486, 15 467 and 15 389 (Recochip 2). Scarcer ends mapping to the NCR were also cloned and sequenced from control littermates (yellow diamonds). The NCR of mouse mtDNA includes three so-called conserved sequenced boxes (CSBs 1–3), a conserved central domain, and a predicted clover-leaf structure, TAS, which is believed to effect termination of 7S DNA (D-loop) synthesis. Three of the ends (15 751, 15 751 and 15 750) in the conserved central domain are flanked by a GC-rich sequence CCGGGCCC and GGGGG, which is extremely rare in the L-strand of mammalian mtDNAs, suggesting that this may represent a cis -element. Free 5′ ends of DNA identified previously by LM-PCR are represented as vertical blue lines; in panel B the free 5′ ends comprise two clusters, cluster I (O H ) and cluster II ( 12 , 13 , 38 ). The height of the most prominent free end (nt 16 034) was set arbitrarily and the others expressed as a fraction of its height. Panel ( C ) The sequenced Sac I/S1 products with an end mapping close to O L (nt 5160–5191) were nt 4946, 5082, 5133, 5201, 5217, 5255, 5257, 5318, 5477 (red diamonds) in mutator mouse samples and nt 5169, 5193, 5196, 5199, 5244 and 5362 (yellow diamonds) in controls. In humans, free 5′ ends are concentrated in the tRNA Cys gene (C), which is adjacent to O L ( 38 , 39 ) and free 5′ ends map to similar positions in mouse mtDNA (see Supplementary Figure 2 ); in panel (C) they are again represented as blue vertical lines. Panel (D) Sac I/S1-treated mutator mouse mtDNA analysed by 1D-AGE, iii is the material that was used to define the ends of DNA represented as red diamonds in panel (C).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Amplification, Clone Assay, Sequencing, Polymerase Chain Reaction

    Linear 11 kb fragments of mtDNA are present at high abundance in mutator mouse liver mtDNA, in the absence of single-strand nuclease treatment. Liver mtDNA samples isolated from wild-type (Wt) and mutator (M) mice were digested with restriction enzymes, Xho I, Mlu I or Sac I, or left uncut, and fractionated by 1D-AGE in TBE buffer. Separation conditions were 55 V for 20 h in 0.4% agarose (Panels A and B ) or 100 V for 4 h in 0.8% agarose (Panels C and D ). After Southern blotting, membranes were probed with PCR products corresponding to nt 14 903–15 401 (Panels A and C); and nt 8031–8625 (Panels B and D) of mouse mtDNA. The samples in panels (C) and (D) were treated additionally with single-strand specific (S1) nuclease after restriction digestion (see ‘Materials and Methods’ section). The two lanes in panel (C) are different exposures of the same gel; a longer exposure of the digest of Wt mtDNA was needed to show that S1 nuclease generated some fragments of similar size to the abundant short fragments seen in mutator mouse mtDNA digests. The species that distinguished mutator mouse mtDNA from wild-type mtDNA are interpreted as follows: 1 —replicating theta structures, or eyebrows, as illustrated at the base of the figure. Wild-type mtDNA samples also include theta structures but their low abundance means they are difficult to detect unless 2D-AGE is applied ( 12 ); 2 —linear mtDNA fragments of approximately 11 kb; 3 — Xho I digested mtDNA fragments with one end corresponding to the restriction site at nt 13 558 and the other mapping to the major non-coding region (NCR); 4 — Sac I fragments ∼7 kb, spanning nt 9047 to the NCR; 5 —fragments ∼8 kb, with one end close to O L the other nt 13 558; 6 —fragments ∼4 kb, with termini at nt 9047 and near O L . The fragments labelled 7 in panel D were gel-extracted, converted to circles, cloned and sequenced, without recourse to S1 nuclease treatment, and found to contain point mutations, which indicated that the novel fragments were the result of Sac I site gains (see Supplementary Figure 3 for details).

    Journal: Nucleic Acids Research

    Article Title: Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    doi: 10.1093/nar/gkp091

    Figure Lengend Snippet: Linear 11 kb fragments of mtDNA are present at high abundance in mutator mouse liver mtDNA, in the absence of single-strand nuclease treatment. Liver mtDNA samples isolated from wild-type (Wt) and mutator (M) mice were digested with restriction enzymes, Xho I, Mlu I or Sac I, or left uncut, and fractionated by 1D-AGE in TBE buffer. Separation conditions were 55 V for 20 h in 0.4% agarose (Panels A and B ) or 100 V for 4 h in 0.8% agarose (Panels C and D ). After Southern blotting, membranes were probed with PCR products corresponding to nt 14 903–15 401 (Panels A and C); and nt 8031–8625 (Panels B and D) of mouse mtDNA. The samples in panels (C) and (D) were treated additionally with single-strand specific (S1) nuclease after restriction digestion (see ‘Materials and Methods’ section). The two lanes in panel (C) are different exposures of the same gel; a longer exposure of the digest of Wt mtDNA was needed to show that S1 nuclease generated some fragments of similar size to the abundant short fragments seen in mutator mouse mtDNA digests. The species that distinguished mutator mouse mtDNA from wild-type mtDNA are interpreted as follows: 1 —replicating theta structures, or eyebrows, as illustrated at the base of the figure. Wild-type mtDNA samples also include theta structures but their low abundance means they are difficult to detect unless 2D-AGE is applied ( 12 ); 2 —linear mtDNA fragments of approximately 11 kb; 3 — Xho I digested mtDNA fragments with one end corresponding to the restriction site at nt 13 558 and the other mapping to the major non-coding region (NCR); 4 — Sac I fragments ∼7 kb, spanning nt 9047 to the NCR; 5 —fragments ∼8 kb, with one end close to O L the other nt 13 558; 6 —fragments ∼4 kb, with termini at nt 9047 and near O L . The fragments labelled 7 in panel D were gel-extracted, converted to circles, cloned and sequenced, without recourse to S1 nuclease treatment, and found to contain point mutations, which indicated that the novel fragments were the result of Sac I site gains (see Supplementary Figure 3 for details).

    Article Snippet: Xho I/S1 treated mouse mtDNA was recovered from 1D agarose gels by electrophoresis at 70 V for 2 h into Recochips™ (TaKaRa, Japan) and ligated overnight with 200 U of T4 ligase (New England Biolabs), at 22°C; the junction of the ligated DNA fragments was amplified over the course of 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 30 s using Biotaq (Bioline), or KOD HiFi DNA polymerase (Novagen).

    Techniques: Isolation, Mouse Assay, Southern Blot, Polymerase Chain Reaction, Generated, Clone Assay

    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

    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