Overlap Extension PCR. The ihpRNA structures were generated by Overlap Extension PCR in 1 tube. The 50 μl reactions contained 0.1–1 ng plasmid DNA or purified PCR product for each template (templates for stem and intron), 400 nM FP, 20 nM of each BP (BP1–4), 200 μM dNTPs, 1× LA Taq Buffer (Mg2+Plus), and 1.25–2.5 U LA Taq. The reactions started at 95 °C for 4 min, followed by 40 cycles of 95 °C for 30 s, 46–56 °C (depending on the Tm of FP, Table 1) for 30 s, and 72 °C for 60–90 s (depending on the length of ihpRNA), and
Sunitinib Malate final extension at 72 °C for 5 min. To optimize the asymmetric PCR, 6 different concentrations (400, 80, 40, 20, 10 and 4 nM) of the BPs (BP1–4) were added, along with 400 nM FP2. To determine the optimum anneal temperature, various anneal temperatures (46.2, 47.3, 49.2, 41.9, 55.7, 58.4, 60.2 and 61.2 °C) between Tm ?20 and Tm ?5 °C (the Tm of FP2) were used to perform OE-PCR. To test the effect of different DNA Polymerase on the PCR process, 5 types of DNA Polymerase were purchased from 3 companies and were used to amplify the ihpRNA, H2, at a
cast BP to FP ratio of 1:20.
