CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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25 The samples were mixed with 6X non-denaturing loading dye buffer. Before sample loading, the gel was pre-run at 350 votes for 30 min at 4 o C in 1X TBE buffer supplemented with 50mM KCl. Each well was washed again and 10 µl of each sample was loaded. After all samples were loaded, the gel was run at 350 votes at 4 o C until the dye front traveled about 10 cm from the wells. Then, the power supply was shut off and the glass wash, ready for fluorescence detection. 2.5 Denaturing gel electrophoresis Denaturing gel electrophoresis was employed to separate DNA by length. In this study, we employed this technique to separate the DNA products from DNA polymerase stop assay. The gel mold was prepared as described. The denaturing gel was mixing 40% acrylamide/bis-acrylamide (19:1), urea, 5X TBE buffer, and the water to the desired percentage of gel and the final concentration of 8 M urea. For 80 ml gel, the gel polymerization was started by adding 1ml of 10% ammonium persulfate and 35 µl of TEMED before pouring to the gel mold. When polymerization was complete, the gel plate was assembled to electrophoresis unit. The gel slots were washed and the gel was pre-run at constant power 1800-2000 voltage for 1 hour. Once again, slots were washed to eliminate the accumulated urea. Before sample loading, the sample in formamide loading buffer were heated at 95 O C for 5 min and immediately placed on ice, to denature any secondary structure. Samples were then loaded onto the gel and electrophoresis in a 1X TBE running buffer at 1800 volts for 2 hours. The gel was separated from the glass plates and transferred onto a plastic covered, ready to detect fluorescence bands.
2.6 DNA polymerase stop assay 26 DNA polymerase stop assay is often employed to test whether a compound can induce intramolecular G-quadruplex formation from any particular G-quadruplex motif. The assay is based on the principle that DNA polymerase cannot traverse through an intramolecular G-quadruplex structure; therefore, primer extension by DNA polymerase will arrest when DNA polymerase encounters a stable G- quadruplex on the DNA template. The 10 l G-quadruplex forming mixture, consisting of the indicated concentration of test compound, a DNA template (VT-66, 100 nM), and a FAM-labeled primer (P16, 100 nM) in 10 mM Tris-HCl buffer (pH 7.4) containing 25 mM KCl, was first denatured by heating to 95 C for 5 min, then slowly cooled to 37 C, and held at 37 C for 30 min. The primer extension reaction was initiated by adding a 10 l reaction mixture consisting of 100 mM dNTPs, 3 mM MgCl2, and 1 unit of DNA polymerase I Klenow fragment (Fermentas). The reaction was allowed to proceed at 37 C for 30 min before it was stopped by adding 4 l of loading buffer (95% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue). The samples were separated by electrophoresis in a 12% denaturing polyacrylamide gel and visualized with a phosphoimaging system (Typhoon; Molecular Dynamics). 2.7 Duplex/quadruplex competition Assay Many G-quadruplex ligands are not useful as telomerase inhibitor because they induce acute cytotoxicity, which is believed to occur from non-specific binding to double stranded DNA. To investigate the binding preference of our perylene derivatives, we employed the duplex/quadruplex competition assay. If the compound
Page 1 and 2: 2.1 Chemicals and materials CHAPTER
Page 3: Name 24 Table 2.2: Sequences of oli
Page 7 and 8: 2.9 Cytotoxicity test 28 Cell survi
Page 9 and 10: 30 with 5% CO2. The first plate, as
Page 11 and 12: 32 Table 2.3 Primer sequences for d
Page 13 and 14: 34 2.10.3 cDNA synthesis by reverse
Page 15 and 16: 36 2.11.1 Preparation of total cell
Page 17 and 18: 38 through polyacrylamide gel. Smal

CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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