Reverse primer 28s cl r

Each PCR amplification with pfu DNA polymerase (Agilent, Santa Clara, CA) alone or combined with DreamTaq™ was carried out in a 25 μl of mixture containing pfu (2.5 units/μl) 0.75 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10× Pfu reaction buffer, 10× PicoMaxx™ buffer, or 10× DreamTaq™ buffer 2.5 μl, dNTP (25 mM each) 0.2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water (Sigma-Aldrich, St Louis, MO) 16.85 μl (or 16.725 μl). The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 55°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.

Each PCR amplification with PicoMaxx™ High Fidelity PCR System (PicoMaxx™ System) (Agilent, Santa Clara, CA) alone or combined with DreamTaq™ was carried out in a 25 μl of mixture containing PicoMaxx™ high fidelity PCR system (PicoMaxx™ (5 units/μl)) 0.5 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10× PicoMaxx™ reaction buffer (PicoMaxx™ buffer) 2.5 μl, dNTP (25 mM each) 0.2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water (Sigma-Aldrich, St Louis, MO) 17.3 μl (or 17.175 μl). The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 55°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.