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  • 93
    TaKaRa exonuclease iii
    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease <t>III.</t> To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the <t>DNA</t> was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
    Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa exonuclease iii buffer
    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the <t>pGL2-IL-6</t> deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among <t>three</t> separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.
    Exonuclease Iii Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    TaKaRa exonucleases iii
    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the <t>pGL2-IL-6</t> deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among <t>three</t> separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.
    Exonucleases Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    exonucleases iii - by Bioz Stars, 2020-08
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    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Journal: Molecular and Cellular Biology

    Article Title: In Vitro Reconstitution of the End Replication Problem

    doi: 10.1128/MCB.21.17.5753-5766.2001

    Figure Lengend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).

    Techniques: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay

    The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the pGL2-IL-6 deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among three separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.

    Journal: Cancer research

    Article Title: A GALECTIN-3-DEPENDENT PATHWAY UPREGULATES INTERLEUKIN-6 IN THE MICROENVIRONMENT OF HUMAN NEUROBLASTOMA

    doi: 10.1158/0008-5472.CAN-11-2165

    Figure Lengend Snippet: The distal region of the IL-6 promoter contains functional C/EBP motifs A. IL-6 promoter Luciferase activity in PC3 cells transfected with the pGL2-IL-6 deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among three separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.

    Article Snippet: IL-6 promoter deletion mutants in the pGL2-IL-6-Luc construct were generated either by Exonuclease III digestion using the Deletion Kit for kilo sequencing (Takara) in accordance with the instructions of the manufacturer (deletion −1041) or by restriction endonuclease digestion with KpnI and NheI (deletion mutant −212) followed by Klenow fragment reaction at 37°C for 15 minutes.

    Techniques: Functional Assay, Luciferase, Activity Assay, Transfection, Sequencing

    Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Journal: FEBS Open Bio

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes

    doi: 10.1016/j.fob.2013.08.007

    Figure Lengend Snippet: Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Article Snippet: Briefly, each reconstituted nucleosome, containing tlH2A, tlH2B, tlH3, or tlH4, was treated with 5 units of Escherichia coli exonuclease III (Takara), in 10 μl of 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2 , and 1 mM DTT.

    Techniques: Purification, Polyacrylamide Gel Electrophoresis

    The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

    Journal: Scientific Reports

    Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

    doi: 10.1038/srep07115

    Figure Lengend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

    Article Snippet: MNase and exonuclease III treatment assays The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes (0.4 μM) were treated with MNase (Takara) or ExoIII (Takara).

    Techniques: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

    Stability of the CENP-A/H3.3 nucleosome. (a) Schematic representation of the thermal stability assay. (b) Thermal stability curve of the H3.3 nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the H3.3 stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (c) A thermal stability curve of the CENP-A nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the CENP-A stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (d) Tetrasomes, reconstituted with the H3.3-H4 tetramer or the CENP-A-H4 tetramer and DNA, were analyzed by 6% native PAGE. Lane 1 indicates the H3.3 tetrasome before incubation. Lanes 2, 3, 4, 5, and 6 indicate the H3.3 tetrasomes after 25°C, 35°C, 45°C, 55°C, and 65°C incubations, respectively. Lane 7 indicates the CENP-A tetrasome before incubation. Lanes 8, 9, 10, 11, and 12 indicate the CENP-A tetrasomes after 25°C, 35°C, 45°C, 55°C, and 65°C incubations, respectively. DNA was visualized by ethidium bromide staining. The gel image is a representative of seven independent experiments with similar results. (e) A thermal stability curve of the CENP-A/H3.3 nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the CENP-A/H3.3 stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (f) The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were incubated for 1 min at each temperature from 25°C, and the samples at 65°C, 72°C, 79°C, and 86°C were analyzed by 6% native PAGE. DNA was visualized by ethidium bromide staining. The gel image is a representative of three independent experiments with similar results.

    Journal: Scientific Reports

    Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

    doi: 10.1038/srep07115

    Figure Lengend Snippet: Stability of the CENP-A/H3.3 nucleosome. (a) Schematic representation of the thermal stability assay. (b) Thermal stability curve of the H3.3 nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the H3.3 stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (c) A thermal stability curve of the CENP-A nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the CENP-A stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (d) Tetrasomes, reconstituted with the H3.3-H4 tetramer or the CENP-A-H4 tetramer and DNA, were analyzed by 6% native PAGE. Lane 1 indicates the H3.3 tetrasome before incubation. Lanes 2, 3, 4, 5, and 6 indicate the H3.3 tetrasomes after 25°C, 35°C, 45°C, 55°C, and 65°C incubations, respectively. Lane 7 indicates the CENP-A tetrasome before incubation. Lanes 8, 9, 10, 11, and 12 indicate the CENP-A tetrasomes after 25°C, 35°C, 45°C, 55°C, and 65°C incubations, respectively. DNA was visualized by ethidium bromide staining. The gel image is a representative of seven independent experiments with similar results. (e) A thermal stability curve of the CENP-A/H3.3 nucleosome. The fluorescence intensity was plotted against each temperature (from 55°C to 90°C). The derivative values of the CENP-A/H3.3 stability curve presented in the upper panel are plotted against the temperatures (bottom panel). Means ± s.d. ( n = 3) are shown. (f) The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were incubated for 1 min at each temperature from 25°C, and the samples at 65°C, 72°C, 79°C, and 86°C were analyzed by 6% native PAGE. DNA was visualized by ethidium bromide staining. The gel image is a representative of three independent experiments with similar results.

    Article Snippet: MNase and exonuclease III treatment assays The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes (0.4 μM) were treated with MNase (Takara) or ExoIII (Takara).

    Techniques: Stability Assay, Fluorescence, Clear Native PAGE, Incubation, Staining

    CENP-C binding to the CENP-A/H3.3 nucleosome. (a) Schematic representations of CENP-C binding to the H3.3 nucleosome, the CENP-A nucleosome, and the CENP-A/H3.3 nucleosome. (b) The binding of CENP-C to the CENP-A/H3.3 nucleosome. CENP-C(426–537) peptide binding to the H3.3 nucleosome (0.2 μM), the CENP-A nucleosome (0.2 μM), and the CENP-A/H3.3 nucleosome (0.2 μM) was evaluated by a gel mobility shift assay. The CENP-C(426–537) peptide concentrations are 0 μM (lanes 1, 3, and 10), 0.2 μM (lanes 4 and 11), 0.4 μM (lanes 5 and 12), 0.6 μM (lanes 6 and 13), 0.8 μM (lanes 2, 7, and 14), 1.0 μM (lanes 8 and 15), and 1.2 μM (lanes 9 and 16). The gel image is a representative of three independent experiments with similar results.

    Journal: Scientific Reports

    Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

    doi: 10.1038/srep07115

    Figure Lengend Snippet: CENP-C binding to the CENP-A/H3.3 nucleosome. (a) Schematic representations of CENP-C binding to the H3.3 nucleosome, the CENP-A nucleosome, and the CENP-A/H3.3 nucleosome. (b) The binding of CENP-C to the CENP-A/H3.3 nucleosome. CENP-C(426–537) peptide binding to the H3.3 nucleosome (0.2 μM), the CENP-A nucleosome (0.2 μM), and the CENP-A/H3.3 nucleosome (0.2 μM) was evaluated by a gel mobility shift assay. The CENP-C(426–537) peptide concentrations are 0 μM (lanes 1, 3, and 10), 0.2 μM (lanes 4 and 11), 0.4 μM (lanes 5 and 12), 0.6 μM (lanes 6 and 13), 0.8 μM (lanes 2, 7, and 14), 1.0 μM (lanes 8 and 15), and 1.2 μM (lanes 9 and 16). The gel image is a representative of three independent experiments with similar results.

    Article Snippet: MNase and exonuclease III treatment assays The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes (0.4 μM) were treated with MNase (Takara) or ExoIII (Takara).

    Techniques: Binding Assay, Mobility Shift