Telomerase Primer Extension Assay Protocol

Telomerase primer extension assays were performed in 20 μl reactions containing 50 mM Tris-acetate pH 8.0, 4 mM MgCl₂, 5 mM DTT, 250 μM dTTP, 250 μM dATP, 5 μM unlabelled dGTP, 0.1 μM α-^{32 (link)}P dGTP (3000 Ci/mmole, 10 mCi/ml, Perkin Elmer), 500 nM (T₂AG₃)₃. The reactions were performed at 30 °C for 40 min, stopped with 50 mM Tris HCl pH 7.5, 20 mM EDTA, 0.2 % SDS,extracted with phenol:chloform:isoamyl alcohol and precipitated with ethanol and a 12-nt ^{32 (link)}P labelled (T₂AG₃)₂ oligonucleotide. The products were resolved on a 10.5% denaturing polyacrylamide TBE gel, which was dried and exposed to a phosphorimager screen. The screen was scanned using an Amersham Typhoon Biomolecular Imager (Cytiva). Bands were quantified using ImageQuant (Cytiva).