Structured Review

TaKaRa puc18
Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids <t>pUC18</t> (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.
Puc18, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 40 article reviews
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puc18 - by Bioz Stars, 2020-05
92/100 stars

Images

1) Product Images from "BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm"

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

Journal: Retrovirology

doi: 10.1186/1742-4690-7-91

Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.
Figure Legend Snippet: Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.

Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Standard Deviation

2) Product Images from "Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus"

Article Title: Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

Journal: Journal of Bacteriology

doi: 10.1128/JB.01344-12

(A) 5′-RACE analysis of sag SA . Total RNA from early-logarithmic-phase cells was prepared and used for 5′-RACE analysis. (A) The resulting DNA fragments containing the 5′ terminus of cDNA from mRNA sequence(s) of sag SA were amplified by nested PCR and analyzed by agarose-gel electrophoresis. M, hundred-base-pair ladder marker. (B) Cloned 5′-RACE products for sequencing. The 5′-RACE product cloned in the SmaI site of pUC18 was obtained by double digestion with EcoRI and SalI and separated by agarose gel electrophoresis. M, hundred-base-pair ladder size marker. Lanes 1 to 4 show the 5′-end part of cDNA deduced to be the transcripts from sagA1p (lane 1), sagA2p (lanes 2 and 3), and sagBp (lane 4).
Figure Legend Snippet: (A) 5′-RACE analysis of sag SA . Total RNA from early-logarithmic-phase cells was prepared and used for 5′-RACE analysis. (A) The resulting DNA fragments containing the 5′ terminus of cDNA from mRNA sequence(s) of sag SA were amplified by nested PCR and analyzed by agarose-gel electrophoresis. M, hundred-base-pair ladder marker. (B) Cloned 5′-RACE products for sequencing. The 5′-RACE product cloned in the SmaI site of pUC18 was obtained by double digestion with EcoRI and SalI and separated by agarose gel electrophoresis. M, hundred-base-pair ladder size marker. Lanes 1 to 4 show the 5′-end part of cDNA deduced to be the transcripts from sagA1p (lane 1), sagA2p (lanes 2 and 3), and sagBp (lane 4).

Techniques Used: Sequencing, Amplification, Nested PCR, Agarose Gel Electrophoresis, Marker, Clone Assay

3) Product Images from "Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid"

Article Title: Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00737-13

Scheme for vector pZGX04 construction. A large fragment containing orf2 to orf10 was obtained from pZGX01 by digestion with EcoRI and BamHI, and the fragment was ligated to EcoRI-BamHI-digested E. coli plasmid pUC18. The resulting plasmid was digested
Figure Legend Snippet: Scheme for vector pZGX04 construction. A large fragment containing orf2 to orf10 was obtained from pZGX01 by digestion with EcoRI and BamHI, and the fragment was ligated to EcoRI-BamHI-digested E. coli plasmid pUC18. The resulting plasmid was digested

Techniques Used: Plasmid Preparation

4) Product Images from "Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis"

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-018-1309-3

Acyl-CoA thioesterase activity of ptTES1. a ptTES1 was expressed in frame with lacZ (upper) in E. coli K27 fadD mutant fadD88 and resulted in the color change of E. coli colonies grown on M9 agar plate containing neutral red. Colonies that express an active ptTES1 are dark pink, while those containing empty vector pUC18 or expressing palmitoyl protein thioesterase ptPPT1 ( J10454 ) are light yellow. The time-dependent hydrolysis by ptTES1 of varied concentrations of decanoyl-CoA (10:0), myristoyl-CoA (14:0), and stearoyl-CoA (18:0) ( b ); palmitoyl-CoA (16:0), oleoyl-CoA (18:1), and eicosapentaenoyl-CoA (20:5) ( c ); and malonyl-CoA (3:0) ( d ); was monitored by measuring the change of A 420 . Reactions were carried out at 37 °C in mixture including 10 mM Hepes (pH 7.5), 50 mM KCl, and 0.3 mM DTNB
Figure Legend Snippet: Acyl-CoA thioesterase activity of ptTES1. a ptTES1 was expressed in frame with lacZ (upper) in E. coli K27 fadD mutant fadD88 and resulted in the color change of E. coli colonies grown on M9 agar plate containing neutral red. Colonies that express an active ptTES1 are dark pink, while those containing empty vector pUC18 or expressing palmitoyl protein thioesterase ptPPT1 ( J10454 ) are light yellow. The time-dependent hydrolysis by ptTES1 of varied concentrations of decanoyl-CoA (10:0), myristoyl-CoA (14:0), and stearoyl-CoA (18:0) ( b ); palmitoyl-CoA (16:0), oleoyl-CoA (18:1), and eicosapentaenoyl-CoA (20:5) ( c ); and malonyl-CoA (3:0) ( d ); was monitored by measuring the change of A 420 . Reactions were carried out at 37 °C in mixture including 10 mM Hepes (pH 7.5), 50 mM KCl, and 0.3 mM DTNB

Techniques Used: Activity Assay, Mutagenesis, Plasmid Preparation, Expressing

5) Product Images from "Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414"

Article Title: Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414

Journal: Applied and Environmental Microbiology

doi:

Plasmids pGADegl3, pCLegl3, and pAGegl3. Relevant gene locations are indicated. See the text for details on the construction of plasmids. ADH1p and ADH1t , promoter and terminator of the S. cerevisiae ADH1 gene, respectively; LEU2 , LEU2 gene of S. cerevisiae ; 2μ ori, origin of replication of the 2μm plasmid; pUC ori, replication origin of the E. coli pUC18 plasmid; hCMVp, promoter of the hCMV gene; SV40p and SV40t, promoter and terminator of the simian virus 40 (SV40) gene, respectively; neo r , neomycin resistance gene of Tn 5 conferring G418 resistance in Schizosaccharomyces pombe ; pBR ori, origin of replication of the E. coli pBR322 plasmid; Ptac , tac promoter; Trrn , rrnB terminator; Amp r , ampicillin resistance gene.
Figure Legend Snippet: Plasmids pGADegl3, pCLegl3, and pAGegl3. Relevant gene locations are indicated. See the text for details on the construction of plasmids. ADH1p and ADH1t , promoter and terminator of the S. cerevisiae ADH1 gene, respectively; LEU2 , LEU2 gene of S. cerevisiae ; 2μ ori, origin of replication of the 2μm plasmid; pUC ori, replication origin of the E. coli pUC18 plasmid; hCMVp, promoter of the hCMV gene; SV40p and SV40t, promoter and terminator of the simian virus 40 (SV40) gene, respectively; neo r , neomycin resistance gene of Tn 5 conferring G418 resistance in Schizosaccharomyces pombe ; pBR ori, origin of replication of the E. coli pBR322 plasmid; Ptac , tac promoter; Trrn , rrnB terminator; Amp r , ampicillin resistance gene.

Techniques Used: Plasmid Preparation

6) Product Images from "Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance"

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

Journal: Nature Communications

doi: 10.1038/s41467-017-02304-7

Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p
Figure Legend Snippet: Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p

Techniques Used: Mouse Assay, Multiplex Assay, Two Tailed Test

Related Articles

Clone Assay:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster
Article Snippet: .. Fragments 2 and 3 were connected by a fragment-primed PCR procedure and cloned between the Bam HI and Hin dIII sites of pUC18, resulting in 23-pUC18. .. Fragments 5 and 6 were also connected by a fragment-primed PCR procedure and cloned between the Hin dIII and Bam HI sites of pUC18, resulting in 56-pUC18.

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega). .. Calculation of copy number by the serial dilution method pBLV-LTR/SK and pBoLA-DRA/SK were digested with Sca I and purified using a Sephadex G-50 column (GE Healthcare Japan, Tokyo, Japan).

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ). ..

Article Title: Construction of a High-Resolution 2.5-Mb Transcript Map of the Human 6p21.2-6p21.3 Region Immediately Centromeric of the Major Histocompatibility Complex
Article Snippet: .. The DNA was extracted using electrophoresis in DEAE–cellulose membrane , subcloned into appropriately prepared pGEM-5Zf, pGEM7-Zf (Promega), and pUC18 (TaKaRa) vectors, and multiple clones isolated and stored at −80°C. ..

DNA Ligation:

Article Title: Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus
Article Snippet: .. Subsequently, the purified dephosphorylated pUC18 and each phosphorylated 5′-RACE product fraction were ligated using DNA ligation Mighty mix (TaKaRa). .. The competent cells of E. coli JM109 were transformed by each recombinant plasmid and screened on an LB plate containing 50 to 100 μg/ml of ampicillin, 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG), and 0.004% (wt/vol) 5-bromo-4-chloro-3-indolyl-β- d -galactoside (X-Gal).

Synthesized:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Isolation:

Article Title: Construction of a High-Resolution 2.5-Mb Transcript Map of the Human 6p21.2-6p21.3 Region Immediately Centromeric of the Major Histocompatibility Complex
Article Snippet: .. The DNA was extracted using electrophoresis in DEAE–cellulose membrane , subcloned into appropriately prepared pGEM-5Zf, pGEM7-Zf (Promega), and pUC18 (TaKaRa) vectors, and multiple clones isolated and stored at −80°C. ..

Construct:

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ). ..

Purification:

Article Title: Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus
Article Snippet: .. Subsequently, the purified dephosphorylated pUC18 and each phosphorylated 5′-RACE product fraction were ligated using DNA ligation Mighty mix (TaKaRa). .. The competent cells of E. coli JM109 were transformed by each recombinant plasmid and screened on an LB plate containing 50 to 100 μg/ml of ampicillin, 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG), and 0.004% (wt/vol) 5-bromo-4-chloro-3-indolyl-β- d -galactoside (X-Gal).

Electrophoresis:

Article Title: Construction of a High-Resolution 2.5-Mb Transcript Map of the Human 6p21.2-6p21.3 Region Immediately Centromeric of the Major Histocompatibility Complex
Article Snippet: .. The DNA was extracted using electrophoresis in DEAE–cellulose membrane , subcloned into appropriately prepared pGEM-5Zf, pGEM7-Zf (Promega), and pUC18 (TaKaRa) vectors, and multiple clones isolated and stored at −80°C. ..

Polymerase Chain Reaction:

Article Title: Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster
Article Snippet: .. Fragments 2 and 3 were connected by a fragment-primed PCR procedure and cloned between the Bam HI and Hin dIII sites of pUC18, resulting in 23-pUC18. .. Fragments 5 and 6 were also connected by a fragment-primed PCR procedure and cloned between the Hin dIII and Bam HI sites of pUC18, resulting in 56-pUC18.

Plasmid Preparation:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega). .. Calculation of copy number by the serial dilution method pBLV-LTR/SK and pBoLA-DRA/SK were digested with Sca I and purified using a Sephadex G-50 column (GE Healthcare Japan, Tokyo, Japan).

Article Title: Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid
Article Snippet: .. To determine the complete nucleotide sequence of pZGX01, the plasmid was linearized by BamHI and subcloned in pUC18 for sequencing (fold coverage, 30×), which was performed by TaKaRa (Dalian, China). .. The sequencing data were assembled and analyzed by using Vector NTI (version 11; Invitrogen, New York, NY), GLIMMER (version 3.2; Center for Bioinformatics and Computational Biology, University of Maryland [ ]), Promoter 2.0 , GeneMarkS (version 4.7; Georgia Institute of Technology, Atlanta, GA [ ]), and BLAST ( ) software.

Article Title: Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414
Article Snippet: .. Plasmids pKK223-3 (Pharmacia) and pUC18 (Takara Shuzo, Kyoto, Japan) were used for the construction of the E. coli expression vector. pUC18 was digested with Pvu II to eliminate the fragment including the lac promoter, and the remaining fragment was ligated with the DNA fragment obtained after digestion of pKK223-3 with Pvu II that contains the tac promoter and the rrn terminator, to lead to substitution of the multiple-cloning site derived from pKK223-3 for that from pUC18. .. The resulting plasmid, named pAG9-3, was used as an E. coli expression vector.

Expressing:

Article Title: Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414
Article Snippet: .. Plasmids pKK223-3 (Pharmacia) and pUC18 (Takara Shuzo, Kyoto, Japan) were used for the construction of the E. coli expression vector. pUC18 was digested with Pvu II to eliminate the fragment including the lac promoter, and the remaining fragment was ligated with the DNA fragment obtained after digestion of pKK223-3 with Pvu II that contains the tac promoter and the rrn terminator, to lead to substitution of the multiple-cloning site derived from pKK223-3 for that from pUC18. .. The resulting plasmid, named pAG9-3, was used as an E. coli expression vector.

Sequencing:

Article Title: Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid
Article Snippet: .. To determine the complete nucleotide sequence of pZGX01, the plasmid was linearized by BamHI and subcloned in pUC18 for sequencing (fold coverage, 30×), which was performed by TaKaRa (Dalian, China). .. The sequencing data were assembled and analyzed by using Vector NTI (version 11; Invitrogen, New York, NY), GLIMMER (version 3.2; Center for Bioinformatics and Computational Biology, University of Maryland [ ]), Promoter 2.0 , GeneMarkS (version 4.7; Georgia Institute of Technology, Atlanta, GA [ ]), and BLAST ( ) software.

Derivative Assay:

Article Title: Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414
Article Snippet: .. Plasmids pKK223-3 (Pharmacia) and pUC18 (Takara Shuzo, Kyoto, Japan) were used for the construction of the E. coli expression vector. pUC18 was digested with Pvu II to eliminate the fragment including the lac promoter, and the remaining fragment was ligated with the DNA fragment obtained after digestion of pKK223-3 with Pvu II that contains the tac promoter and the rrn terminator, to lead to substitution of the multiple-cloning site derived from pKK223-3 for that from pUC18. .. The resulting plasmid, named pAG9-3, was used as an E. coli expression vector.

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  • 85
    TaKaRa supercoiled puc18 plasmid dna
    The binding of cc-dsDNA to Mhr1 and the topological status of Mhr1-bound cc-dsDNA. A , the binding of cc-dsDNA to Mhr1. Negatively <t>supercoiled</t> <t>pUC18</t> plasmid <t>DNA</t> (41.8 μ m ) was mixed with the indicated concentrations of Mhr1 in standard buffer
    Supercoiled Puc18 Plasmid Dna, 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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    TaKaRa materials plasmid puc19 dna
    Histograms and electropherograms (the right side figure) representing cleavage of <t>pUC19</t> plasmid <t>DNA</t> (0.008 µg/µL) by different concentrations of 1b (pH = 7.4) and 1c (pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 1b ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( B ) Complex 1c ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively.
    Materials Plasmid Puc19 Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    TaKaRa puc18 dna
    Topological inactivation activities. Lane 1 indicated <t>pUC18</t> as a control; Lane 2 indicated the products of pUC18 <t>DNA</t> treated with α-MMC; Lane M indicated DNA ladder; Lane 3 indicated the products of pUC18 DNA treated with MAP30.
    Puc18 Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa undigested puc18
    Double stranded DNA end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized <t>pUC18,</t> black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
    Undigested Puc18, 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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    The binding of cc-dsDNA to Mhr1 and the topological status of Mhr1-bound cc-dsDNA. A , the binding of cc-dsDNA to Mhr1. Negatively supercoiled pUC18 plasmid DNA (41.8 μ m ) was mixed with the indicated concentrations of Mhr1 in standard buffer

    Journal: The Journal of Biological Chemistry

    Article Title: Heteroduplex Joint Formation Free of Net Topological Change by Mhr1, a Mitochondrial Recombinase

    doi: 10.1074/jbc.M900023200

    Figure Lengend Snippet: The binding of cc-dsDNA to Mhr1 and the topological status of Mhr1-bound cc-dsDNA. A , the binding of cc-dsDNA to Mhr1. Negatively supercoiled pUC18 plasmid DNA (41.8 μ m ) was mixed with the indicated concentrations of Mhr1 in standard buffer

    Article Snippet: Preparation of Relaxed cc-dsDNA —Partially or fully relaxed cc-dsDNA was prepared by treating negatively supercoiled pUC18 plasmid DNA (209 μ m in nucleotides) with 0.025 units/μl or 0.1 units/μl calf thymus topoisomerase I (Takara Shuzo Co., Kyoto, Japan) in a 20-μl reaction mixture at 37 °C for 30 min.

    Techniques: Binding Assay, Plasmid Preparation

    Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by different concentrations of 1b (pH = 7.4) and 1c (pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 1b ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( B ) Complex 1c ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by different concentrations of 1b (pH = 7.4) and 1c (pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 1b ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( B ) Complex 1c ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in different pH buffer of 2a (6.25 × 10 −7 mol/L), 2b (3.13 × 10 −7 mol/L) and 2d (6.25 × 10 −7 mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 2a; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively. ( B ) Complex 2b and Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 Lane 9 = DNA control, respectively; ( C ) complex 2d; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in different pH buffer of 2a (6.25 × 10 −7 mol/L), 2b (3.13 × 10 −7 mol/L) and 2d (6.25 × 10 −7 mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 2a; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively. ( B ) Complex 2b and Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 Lane 9 = DNA control, respectively; ( C ) complex 2d; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Electropherograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in buffer of different weight ratios of GO to complex 2c (6.25 × 10 −7 mol/L, 5 mM Tris-HCl/10 mM NaCl, pH = 6.0) at 37 °C for 8 h. Lanes 1–7: complex 2c alone, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, respectively and Lane 8 is DNA control with GO (0.67 μg/μL), respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Electropherograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in buffer of different weight ratios of GO to complex 2c (6.25 × 10 −7 mol/L, 5 mM Tris-HCl/10 mM NaCl, pH = 6.0) at 37 °C for 8 h. Lanes 1–7: complex 2c alone, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, respectively and Lane 8 is DNA control with GO (0.67 μg/μL), respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Electropherograms representing condensation and release of pUC19 plasmid DNA (0.008 µg/µL) in buffer of different weight ratios of GO to complex 2c (5 mM Tris-HCl/10 mM NaCl, pH = 7.4) at 37 °C. Lane 1 is DNA control, Lanes 2–8: 2c (6.25 × 10 −5 mol/L), 2c (3.13 × 10 −5 mol/L), 2c (6.25 × 10 −5 mol/L)/GO (0.67 μg/μL), 2c (6.25 × 10 −5 mol/L)/GO (1.34 μg/μL), 2c (3.13 × 10 −5 mol/L)/GO (0.67 μg/μL), 2c (3.13 × 10 −5 mol/L)/GO (1.34 μg/μL), respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Electropherograms representing condensation and release of pUC19 plasmid DNA (0.008 µg/µL) in buffer of different weight ratios of GO to complex 2c (5 mM Tris-HCl/10 mM NaCl, pH = 7.4) at 37 °C. Lane 1 is DNA control, Lanes 2–8: 2c (6.25 × 10 −5 mol/L), 2c (3.13 × 10 −5 mol/L), 2c (6.25 × 10 −5 mol/L)/GO (0.67 μg/μL), 2c (6.25 × 10 −5 mol/L)/GO (1.34 μg/μL), 2c (3.13 × 10 −5 mol/L)/GO (0.67 μg/μL), 2c (3.13 × 10 −5 mol/L)/GO (1.34 μg/μL), respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by different concentrations of 2b (pH = 7.4), 2c (pH = 7.4) and 2d (pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37°C for 6 h. ( A ) Complex 2b ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( B ) Complex 2c; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( C ) Complex 2d ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by different concentrations of 2b (pH = 7.4), 2c (pH = 7.4) and 2d (pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37°C for 6 h. ( A ) Complex 2b ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( B ) Complex 2c; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively; ( C ) Complex 2d ; Lanes 1–6: 6.25 × 10 − 5 , 3.13 × 10 − 5 , 6.25 × 10 − 6 , 3.13 × 10 − 6 , 6.25 × 10 − 7 , 3.13 × 10 − 7 mol/L, Lane 7 = DNA control, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 1b (6.25 × 10 −7 mol/L) in pH = 7.4 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, reaction time 6, 5, 4, 3, 2, 1 h, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 1b (6.25 × 10 −7 mol/L) in pH = 7.4 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, reaction time 6, 5, 4, 3, 2, 1 h, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques:

    Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 2c (3.13 × 10 −7 mol/L) in pH = 6.0 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, 6, 5, 4, 3, 2, 1 h reaction time, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 2c (3.13 × 10 −7 mol/L) in pH = 6.0 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, 6, 5, 4, 3, 2, 1 h reaction time, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques:

    Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 2b (3.13 × 10 −7 mol/L) in pH = 7.4 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, reaction time 6, 5, 4, 3, 2, 1 h, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Time course of pUC19 DNA (0.008 µg/µL) cleavage promoted by 2b (3.13 × 10 −7 mol/L) in pH = 7.4 buffers (5 mM Tris-HCl/10 mM NaCl) at 37 °C. Lanes 1–6, reaction time 6, 5, 4, 3, 2, 1 h, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques:

    Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 2a typical radical scavengers (6.25 × 10 −7 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: DMSO, Lane 6: t -BuOH.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 2a typical radical scavengers (6.25 × 10 −7 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: DMSO, Lane 6: t -BuOH.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1a with different typical radical scavengers (6.25 × 10 −7 mol/L, pH = 8.0). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: DMSO, Lane 6: t -BuOH.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1a with different typical radical scavengers (6.25 × 10 −7 mol/L, pH = 8.0). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: DMSO, Lane 6: t -BuOH.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b typical radical scavengers (3.13 × 10 −4 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: t -BuOH, Lane 6: DMSO.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b typical radical scavengers (3.13 × 10 −4 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor, Lane 3: NaN 3 , Lane 4: KI, Lane 5: t -BuOH, Lane 6: DMSO.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in different pH buffer of 1a (6.25 × 10 −7 mol/L), 1b (3.13 × 10 −6 mol/L) and 1c (6.25 × 10 −7 mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 1a ; Lanes 1−5: pH = 6.5, 7.0, 7.4, 8.0, 8.3, Lane 6 = DNA control, respectively; ( B ) Complex 1b; Lanes 1–7: pH = 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 8 = DNA control, respectively; ( C ) Complex 1c; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively.

    Journal: Molecules

    Article Title: DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide

    doi: 10.3390/molecules21070920

    Figure Lengend Snippet: Histograms and electropherograms (the right side figure) representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) in different pH buffer of 1a (6.25 × 10 −7 mol/L), 1b (3.13 × 10 −6 mol/L) and 1c (6.25 × 10 −7 mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. ( A ) Complex 1a ; Lanes 1−5: pH = 6.5, 7.0, 7.4, 8.0, 8.3, Lane 6 = DNA control, respectively; ( B ) Complex 1b; Lanes 1–7: pH = 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 8 = DNA control, respectively; ( C ) Complex 1c; Lanes 1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively.

    Article Snippet: Materials Plasmid pUC19 DNA (TaKaRa Biotechnology, Dalian, China), 50 × TAE, 6× loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).

    Techniques: Plasmid Preparation

    Topological inactivation activities. Lane 1 indicated pUC18 as a control; Lane 2 indicated the products of pUC18 DNA treated with α-MMC; Lane M indicated DNA ladder; Lane 3 indicated the products of pUC18 DNA treated with MAP30.

    Journal: PLoS ONE

    Article Title: A Novel Method for Simultaneous Production of Two Ribosome-Inactivating Proteins, ?-MMC and MAP30, from Momordica charantia L

    doi: 10.1371/journal.pone.0101998

    Figure Lengend Snippet: Topological inactivation activities. Lane 1 indicated pUC18 as a control; Lane 2 indicated the products of pUC18 DNA treated with α-MMC; Lane M indicated DNA ladder; Lane 3 indicated the products of pUC18 DNA treated with MAP30.

    Article Snippet: LMW Calibration Kit was supplied by SIBAS (Shanghai, China). pUC18 DNA used in detection of topological activity was obtained from TAKARA (Dalian, China).

    Techniques:

    Double stranded DNA end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Double stranded DNA end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Article Snippet: DNA substrate determination Assay mixtures (20 μl) containing SXT-Exo (0.6 μg, 5 pmol of trimers) and a DNA substrate: i) undigested pUC18 (184 ng); ii) PstI-linearized pUC18 (184 ng); iii) dephosphorylated PstI-linearized pUC18 (184 ng); or iv) M13-phage ssDNA (TaKaRa, 300 ng, 0.13 pmole) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl with/without 10 mM MgCl2 (as indicated in the text), were incubated at 37°C for 30 minutes then quenched (20 mM EDTA).

    Techniques: Incubation, Standard Deviation

    Processivity of SXT-Exo digestion of double stranded DNA . Heparin-trap experiments were used to calculate the average number of nucleotides hydrolyzed by an SXT-Exo trimer during a single binding event. SXT-Exo (41 nmol of trimers) and PstI-linearized pUC18 DNA (2686 bp in length; 18.1 pmol) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 s, before trapping unbound protein by addition of a large excess of heparin. Aliquots were removed at 0, 1, 2, 5, 10, 20 and 30 minutes; quenched, then dsDNA levels immediately quantified using PicoGreen assays to determine the number of nucleotides digested from each end (red circles) at each time point. Analogous control experiments without heparin were performed (black circles). Four independent replicates of each experiment were conducted, and graphs show the mean values ± standard deviation. See materials section for details.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Processivity of SXT-Exo digestion of double stranded DNA . Heparin-trap experiments were used to calculate the average number of nucleotides hydrolyzed by an SXT-Exo trimer during a single binding event. SXT-Exo (41 nmol of trimers) and PstI-linearized pUC18 DNA (2686 bp in length; 18.1 pmol) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 s, before trapping unbound protein by addition of a large excess of heparin. Aliquots were removed at 0, 1, 2, 5, 10, 20 and 30 minutes; quenched, then dsDNA levels immediately quantified using PicoGreen assays to determine the number of nucleotides digested from each end (red circles) at each time point. Analogous control experiments without heparin were performed (black circles). Four independent replicates of each experiment were conducted, and graphs show the mean values ± standard deviation. See materials section for details.

    Article Snippet: DNA substrate determination Assay mixtures (20 μl) containing SXT-Exo (0.6 μg, 5 pmol of trimers) and a DNA substrate: i) undigested pUC18 (184 ng); ii) PstI-linearized pUC18 (184 ng); iii) dephosphorylated PstI-linearized pUC18 (184 ng); or iv) M13-phage ssDNA (TaKaRa, 300 ng, 0.13 pmole) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl with/without 10 mM MgCl2 (as indicated in the text), were incubated at 37°C for 30 minutes then quenched (20 mM EDTA).

    Techniques: Binding Assay, Incubation, Standard Deviation

    Optimal pH, temperature and Mg(II) and Mn(II) ion concentrations for the dsDNA exonuclease activities of SXT-Exo, as determined by quenched PicoGreen Assays . Panel A : Optimum Mg 2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MnCl 2 (0-10 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel B : Optimum Mn 2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MgCl 2 (0-50 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel C : Optimum pH. SXT-Exo (2 pmol of trimers) in Tris-HCl (50 mM, adjusted to pH 7.0-9.0), 50 mM NaCl, 0.5 mM MnCl 2 ; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel D : Optimum temperature. SXT-Exo (6 pmol of trimers) in Tris-HCl (25 mM, pH 7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 1 min. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Optimal pH, temperature and Mg(II) and Mn(II) ion concentrations for the dsDNA exonuclease activities of SXT-Exo, as determined by quenched PicoGreen Assays . Panel A : Optimum Mg 2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MnCl 2 (0-10 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel B : Optimum Mn 2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MgCl 2 (0-50 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel C : Optimum pH. SXT-Exo (2 pmol of trimers) in Tris-HCl (50 mM, adjusted to pH 7.0-9.0), 50 mM NaCl, 0.5 mM MnCl 2 ; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel D : Optimum temperature. SXT-Exo (6 pmol of trimers) in Tris-HCl (25 mM, pH 7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 1 min. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Article Snippet: DNA substrate determination Assay mixtures (20 μl) containing SXT-Exo (0.6 μg, 5 pmol of trimers) and a DNA substrate: i) undigested pUC18 (184 ng); ii) PstI-linearized pUC18 (184 ng); iii) dephosphorylated PstI-linearized pUC18 (184 ng); or iv) M13-phage ssDNA (TaKaRa, 300 ng, 0.13 pmole) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl with/without 10 mM MgCl2 (as indicated in the text), were incubated at 37°C for 30 minutes then quenched (20 mM EDTA).

    Techniques: Incubation, Standard Deviation

    Effects of addition of various monovalent and divalent salts on the double strand DNA activities of SXT-Exo, as determined by quenched PicoGreen assays . Panel A: Inhibition of the dsDNA exonuclease activities of SXT-Exo with sodium chloride (black squares), sodium phosphate (buffered to pH7.4; red circles) and sodium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (NaCl, Na 2 HPO 4 (pH7.4), or Na 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Panel B: Inhibition of the dsDNA exonuclease activities of SXT-Exo with potassium chloride (black squares), calcium chloride (red squares) and potassium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (KCl, CaCl 2 or K 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Relative dsDNA exonuclease activities were calculated (as a percentage) by comparison with results from analogous assays that contained: SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Effects of addition of various monovalent and divalent salts on the double strand DNA activities of SXT-Exo, as determined by quenched PicoGreen assays . Panel A: Inhibition of the dsDNA exonuclease activities of SXT-Exo with sodium chloride (black squares), sodium phosphate (buffered to pH7.4; red circles) and sodium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (NaCl, Na 2 HPO 4 (pH7.4), or Na 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Panel B: Inhibition of the dsDNA exonuclease activities of SXT-Exo with potassium chloride (black squares), calcium chloride (red squares) and potassium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (KCl, CaCl 2 or K 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Relative dsDNA exonuclease activities were calculated (as a percentage) by comparison with results from analogous assays that contained: SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Article Snippet: DNA substrate determination Assay mixtures (20 μl) containing SXT-Exo (0.6 μg, 5 pmol of trimers) and a DNA substrate: i) undigested pUC18 (184 ng); ii) PstI-linearized pUC18 (184 ng); iii) dephosphorylated PstI-linearized pUC18 (184 ng); or iv) M13-phage ssDNA (TaKaRa, 300 ng, 0.13 pmole) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl with/without 10 mM MgCl2 (as indicated in the text), were incubated at 37°C for 30 minutes then quenched (20 mM EDTA).

    Techniques: Inhibition, Incubation, Standard Deviation

    Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P

    Article Snippet: DNA substrate determination Assay mixtures (20 μl) containing SXT-Exo (0.6 μg, 5 pmol of trimers) and a DNA substrate: i) undigested pUC18 (184 ng); ii) PstI-linearized pUC18 (184 ng); iii) dephosphorylated PstI-linearized pUC18 (184 ng); or iv) M13-phage ssDNA (TaKaRa, 300 ng, 0.13 pmole) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl with/without 10 mM MgCl2 (as indicated in the text), were incubated at 37°C for 30 minutes then quenched (20 mM EDTA).

    Techniques: Incubation, Standard Deviation