Thermal cycling conditions and resultant products from first and second rounds of amplification. Panel A illustrates the thermal cycling conditions for the primary and secondary amplifications. During the first round amplification (Panel A, left side) with gene-specific chimeric primers, step-down thermal cycling PCR conditions involve decreasing the annealing temperature by 1°C per cycle beginning at 62°C for 10 cycles and the subsequent 24 cycles have a set annealing temperature of 52°C. The graph on the right depicts the conditions for constant annealing temperature of 55°C for the second round amplification using a common set of adaptamers primers complementary to the entire set of first round products (Panel A, right side). Panel B illustrates one half (48 reactions) of the results from the primary (above) and secondary (below) amplification of one 96-well plate. Lanes 2 and 6 (arrows 1 and 2) illustrate the product 'leveling effect' on the yields of product due to the secondary amplification with adaptamer primers. Lanes 9, 14, and 35 (arrows 3, 4, and 7) illustrate missing or double products which are scored as bad and have new primers designed for subsequent use in another PCR run. Lanes 17 and 34 (under arrows 5 and 6, respectively) contain molecular weight markers.