10.2.8.4. OneStep PCR

Yue and Genersch (2005) developed a modified OneStep protocol for strand-specific RT-PCR that does not include specific steps to remove the tagged cDNA primer prior to PCR. Occasionally, weak bands are produced derived from non-strand-specific cDNA priming events (Gisder et al., 2009).

1. For the OmniScript/SensiScript OneStep RT-PCR reactions, add:
1.1.  0.5 µl     10 µM tag primer             (0.25 µM final concentration),
1.2.  0.5 µl     10 µM virus-specific primer         (0.25 µM final concentration).
2. Incubate  
  2.1.  15 min at 95oC,
  2.2.  35 cycles of [94oC:30 sec – 54.5oC:60 sec – 72oC:30 sec],
  2.3.  72oC:10 min.