In vitro G4-DNA formation and native polyacrylamide gel electrophoresis

Commercially synthesized G4-DNA oligonucleotides (Integrated DNA Technologies) were rehydrated using ultra-pure DNA grade H₂O to a final concentration of 0.25 mM. To form tetramolecular G4-DNA, aliquots of each sample were boiled in a thermalcycler at 98°C for 10 min, and subsequently held at 80°C as previously described (62). Pre-heated KCl solution (folded G4) or an equal volume of pre-heated ultrapure H₂O (unfolded controls) was added to each aliquot. The final concentration of KCl varied from 50 to 250 mM. The samples were then allowed to slowly cool to room temperature and stored at –4°C until needed (max 3 days). The formation of tetramolecular G4-DNA structures was confirmed via gel electrophoresis using 10 and 20% non-denaturing polyacrylamide gel and Tris/boric acid buffer. To visualize the oligonucleotides and differentiate between G4/ssDNA and dsDNA, resulting gels were stained first with ethidium bromide, imaged on UV Transilluminator FBTIV-88 (Fisher Scientific), then re-stained with Thioflavin T and imaged again.