What PCR enzymes can be used following the first-strand synthesis?
For robust PCR amplification of targets (2 kb or less) among the cDNA pool generated in first-strand synthesis, Phoenix Hot Start Taq DNA Polymerase (ENZ P7590) is recommended. For amplification of long PCR targets (from >2 kb to 12 kb), we suggest VeraSeq Ultra DNA Polymerase (ENZ P7520).
Note: Generally, 1–2 µl (5–10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR, although the precise amount of cDNA added may require optimization depending on target abundance. Addition of RNase H (ENZ Y9220) (5 U, incubation at 37°C for 20 minutes, heat kill at 65°C for 10 minutes) to degrade the RNA strand following cDNA synthesis (prior to PCR addition) may increase PCR amplification efficiency for some targets, especially those >1 kb (Kitabayashi, M., et al. (2003) Biosci. Biotechnol. Biochem. 67:2474).
Note: Generally, 1–2 µl (5–10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR, although the precise amount of cDNA added may require optimization depending on target abundance. Addition of RNase H (ENZ Y9220) (5 U, incubation at 37°C for 20 minutes, heat kill at 65°C for 10 minutes) to degrade the RNA strand following cDNA synthesis (prior to PCR addition) may increase PCR amplification efficiency for some targets, especially those >1 kb (Kitabayashi, M., et al. (2003) Biosci. Biotechnol. Biochem. 67:2474).