What PCR enzymes can be used following 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 (> 2 kB, up to 12 kB), we suggest VeraSeq® Ultra DNA Polymerase (ENZ P7520).
Note: Generally, 1 to 2 µl (5 to 10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR reaction, 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*.
*Reference:
Kitabayashi M. et al., Biosci. Biotechnol. Biochem. (2003) 67 (11):2474-6
Note: Generally, 1 to 2 µl (5 to 10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR reaction, 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*.
*Reference:
Kitabayashi M. et al., Biosci. Biotechnol. Biochem. (2003) 67 (11):2474-6