![]() ![]() ![]() Compounds or distilled water were added under a volume of 5 μl. PCR was performed in a final 50-μl reaction volume composed of a 45-μl reaction mix containing 20 m m Tris-HCl (pH 8.0), 50 μ m deoxynucleotide triphosphates, 1.5 m m MgCl 2, 63 m m KCl, 1 m m EGTA, 0.005% Tween 20, 20 μg/ml BSA, 3.5 pmol of primer TSG4 (5′-GGGATTGGGATTGGGATTGGGTT-3′), 18 pmol of primer TS (5′-AATCCGTCGAGCAGAGTT-3′), 22.5 pmol of primer CXext (5′-GTGCCCTTACCCTTACCCTTACCCTAA-3′), 7.5 pmol of primer NT (5′-ATCGCTTCTCGGCCTTTT-3′), 0.01 amol of TSNT internal control (5′-ATTCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′), 2.5 units of Taq DNA polymerase (DyNAzyme II DNA polymerase Ozyme), and 100 ng of telomerase. TRAP-G4 was performed by using a modification of the TRAP assay (12, 13, 14). Telomerase extract was prepared from A549 cells as described before (21). ![]()
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