CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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27 prefers binding to G-quadruplex, there will be less duplex. On the other hand, if the compound prefers binding to duplex, there will be less G-quadruplex formation. In this study, we observed the competition between duplex and quadruplex from the fluorescent-labeled G-rich strand. The 20-µl reaction mixture contained 2µM of fluorescent-labeled G-strand, 10 mM of Tris-HCl buffer pH 7.4 containing 100 mM KCl, and 0-32 µM of perylene derivative. The reaction mixtures were heated at 95 O C for 10 min before incubated at 55 O C for 10 h. The incubation was terminated by rapidly cooling down to 4 O C. The sample were added with 6X glycerol loading dye buffer and kept at 4 O C before loading into non-denaturing gel. The 10 µl of each sample was loaded on 16% non- denaturing polyacrylamide gel containing 50 mM KCl. The gel was run in 1X TBE buffer supplemented with 50 mM KCl at 4 O C, 350 volts for 10 h. Gel was then exposed to fluorescence spectrum by PhosphoImager. 2.8 Cell lines and culture conditions The human lung carcinoma cell line A549 was obtained from American Type Culture Collection (Rockville, MD). The A549 cells were grown in Roswell Park Memorial Institute medium 1640 (RPMI 1640) with 10% fetal bovine serum (FBS) and 1% antibiotics (50 units/ml penicillin, 50 µg/ml streptomycin). The A549 cells were grown as monolayers at 37 2 and 95% air.
2.9 Cytotoxicity test 28 Cell survival is determined by using the colorimetric Sulforhodamine B assay. The assay relies on the ability of Sulforhodamine B to bind to protein components of cells that have been fixed to tissue-culture plates by trichloroacetic acid (TCA). Sulforhodamine B is bright-pink aminoxanthane dye with two sulfonic groups (Figure 2.2) that bind to basic amino-acid residues under mild acidic conditions, and dissociate under basic conditions. As the binding of Sulforhodamine B is stoichimetric, the amount of dye extracted from stained cells is directly proportional to the cell mass (Vichai and Kirtikara, 2006). Figure 2.2 Structure of Sulforhodamine B
Page 1 and 2: 2.1 Chemicals and materials CHAPTER
Page 3 and 4: Name 24 Table 2.2: Sequences of oli
Page 5: 2.6 DNA polymerase stop assay 26 DN
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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