CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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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.