mcm2 cdna clone tc117321 Search Results


mcm2  (OriGene)
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OriGene mcm2
FIG. 2. In vitro DNA replication with partially purified fraction IA in the presence of polyclonal <t>MCM2</t> antibody (-MCM2). Standard replication reactions (15 l) were performed and contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), Pol (0.4 g), and/or MCM2 antibody (0.1 to 1.0 g). Replication products were subjected to DpnI digestion prior to electrophoresis.
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FIG. 2. In vitro DNA replication with partially purified fraction IA in the presence of polyclonal MCM2 antibody (-MCM2). Standard replication reactions (15 l) were performed and contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), Pol (0.4 g), and/or MCM2 antibody (0.1 to 1.0 g). Replication products were subjected to DpnI digestion prior to electrophoresis.

Journal: Journal of Virology

Article Title: Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

doi: 10.1128/jvi.01968-07

Figure Lengend Snippet: FIG. 2. In vitro DNA replication with partially purified fraction IA in the presence of polyclonal MCM2 antibody (-MCM2). Standard replication reactions (15 l) were performed and contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), Pol (0.4 g), and/or MCM2 antibody (0.1 to 1.0 g). Replication products were subjected to DpnI digestion prior to electrophoresis.

Article Snippet: MCM2 (TC117321) and MCM6 (TC110945) cDNA clones were purchased from Origene.

Techniques: In Vitro, Electrophoresis

FIG. 3. (A) Western blot of a crude Ad-infected cell extract and the partially purified fraction IA using anti-MCM2 antibody. (B) Im- munodepletion of MCM from fraction IA with MCM2 antibody. West- ern blot using MCM2 antibody of the supernatant (S/N) or precipitate (IP) of fraction IA in the absence of antibody (left panel) or the presence of anti-MCM2 antibody (right panel). (C) In vitro DNA replication with the mock-treated or anti-MCM2 antibody immunode- pleted fraction of IA shown in panel B. The supernatant after precip- itation in the absence of antibody (no Ab: IA S/N) or in the presence of anti-MCM2 antibody (-MCM2: IA S/N) was tested, along with the precipitant without antibody added (no Ab: IA IP) or with MCM2 antibody added (-MCM2: IA IP). Standard replication reactions were carried out, and mixtures contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), and/or Pol (0.4 g). Replication products were subjected to DpnI digestion prior to elec- trophoresis.

Journal: Journal of Virology

Article Title: Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

doi: 10.1128/jvi.01968-07

Figure Lengend Snippet: FIG. 3. (A) Western blot of a crude Ad-infected cell extract and the partially purified fraction IA using anti-MCM2 antibody. (B) Im- munodepletion of MCM from fraction IA with MCM2 antibody. West- ern blot using MCM2 antibody of the supernatant (S/N) or precipitate (IP) of fraction IA in the absence of antibody (left panel) or the presence of anti-MCM2 antibody (right panel). (C) In vitro DNA replication with the mock-treated or anti-MCM2 antibody immunode- pleted fraction of IA shown in panel B. The supernatant after precip- itation in the absence of antibody (no Ab: IA S/N) or in the presence of anti-MCM2 antibody (-MCM2: IA S/N) was tested, along with the precipitant without antibody added (no Ab: IA IP) or with MCM2 antibody added (-MCM2: IA IP). Standard replication reactions were carried out, and mixtures contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), and/or Pol (0.4 g). Replication products were subjected to DpnI digestion prior to elec- trophoresis.

Article Snippet: MCM2 (TC117321) and MCM6 (TC110945) cDNA clones were purchased from Origene.

Techniques: Western Blot, Infection, In Vitro

FIG. 4. In vitro DNA replication with purified MCM complexes. The MCM2 preparation was purified using the MCM2 His-tagged recombinant protein, and the MCM6 preparation was purified using the MCM6 His-tagged recombinant protein. Both MCM protein sam- ples were purified by phosphocellulose and Ni-NTA chromatography as described in Methods for MCM purification. Standard replication reactions (15 l) were carried out, and mixtures contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), and/or Pol (0.4 g). The replication products were digested with DpnI prior to electrophoresis.

Journal: Journal of Virology

Article Title: Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

doi: 10.1128/jvi.01968-07

Figure Lengend Snippet: FIG. 4. In vitro DNA replication with purified MCM complexes. The MCM2 preparation was purified using the MCM2 His-tagged recombinant protein, and the MCM6 preparation was purified using the MCM6 His-tagged recombinant protein. Both MCM protein sam- ples were purified by phosphocellulose and Ni-NTA chromatography as described in Methods for MCM purification. Standard replication reactions (15 l) were carried out, and mixtures contained, where indicated, RFC (0.01 g), Rep78 (0.2 g), PCNA (0.4 g), IA (6 g), and/or Pol (0.4 g). The replication products were digested with DpnI prior to electrophoresis.

Article Snippet: MCM2 (TC117321) and MCM6 (TC110945) cDNA clones were purchased from Origene.

Techniques: In Vitro, Recombinant, Chromatography, Electrophoresis

FIG. 6. Level of AAV DNA replication in HEK293 cells in the presence of siRNAs. Three targets (each) for MCM2, MCM6, and Pol were tested in a 24-well format. Twenty picomoles of siRNA, 200 ng of pXX6 (which expresses the Ad helper functions), and 200 ng pSM620 (infectious wild-type AAV plasmid) were transfected into 293 cells. Cells were harvested at 24 h, and low-molecular-weight DNA was extracted by Hirt precipitation and quantitated by Southern blotting using a 32P-labeled AAV DNA probe. The amount of DpnI-resistant monomer duplex DNA was quantitated by phosphorimaging analysis and compared to standard concentrations of pSM620 plasmid DNA. 1, no siRNA; 2, control siRNA (no target); 3 to 5, siRNA for Pol ; 6 to 8, siRNA for MCM6; 9 to 11, siRNA for MCM2. The average from four independent reactions is shown; error bars indicate standard deviations.

Journal: Journal of Virology

Article Title: Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

doi: 10.1128/jvi.01968-07

Figure Lengend Snippet: FIG. 6. Level of AAV DNA replication in HEK293 cells in the presence of siRNAs. Three targets (each) for MCM2, MCM6, and Pol were tested in a 24-well format. Twenty picomoles of siRNA, 200 ng of pXX6 (which expresses the Ad helper functions), and 200 ng pSM620 (infectious wild-type AAV plasmid) were transfected into 293 cells. Cells were harvested at 24 h, and low-molecular-weight DNA was extracted by Hirt precipitation and quantitated by Southern blotting using a 32P-labeled AAV DNA probe. The amount of DpnI-resistant monomer duplex DNA was quantitated by phosphorimaging analysis and compared to standard concentrations of pSM620 plasmid DNA. 1, no siRNA; 2, control siRNA (no target); 3 to 5, siRNA for Pol ; 6 to 8, siRNA for MCM6; 9 to 11, siRNA for MCM2. The average from four independent reactions is shown; error bars indicate standard deviations.

Article Snippet: MCM2 (TC117321) and MCM6 (TC110945) cDNA clones were purchased from Origene.

Techniques: Plasmid Preparation, Transfection, Molecular Weight, Southern Blot, Labeling, Control