RT-PCR of ODN-Tm6 (link)A

ODN-X (2 μL, 4 μM) was mixed with 1 μL of 2 μM 12 nt RT primer 5′-HEX-AGAATCATCGAA and denatured at 70 °C for 5 min, then cooled down slowly to 4 °C (1 °C per 10 s) and hold at 4 °C. After adding 5 U M-MuLV reverse transcriptase (NEB) or 5 U SS III reverse transcriptase (Invitrogen), 2 μL corresponding 5 × buffer (NEB M-MuLV buffer or 5 × FS buffer), 3 μL of 100 μM dNTPs and to the RNA-primer mix (For ODN-Tm^{6 (link)}A, additional 1 μL of 100 mM DTT was added), the RT reactions were carried out at 42 °C for 30 min. Then 30 μL deionized formamide was immediately added to the reaction mixture and heated to 90 °C for 10 min, followed by 20% denaturing PAGE analysis.