PCR using the designed primers. (A) The optimisation of the annealing temperatures. Each hybrid primer was tested for amplification at three different annealing temperatures, roughly chosen as Tm-7°C, Tm-5°C and Tm-3°C (based on Tm values calculated using on-line primer design software from Sigma-Genosys). Hybrid primer M22F (panel A, left gel) does not show any preference for annealing temperature. Primer M15F (panel A, middle) shows strong preference for Tm-7°C. Primer 06F (panel A, right panel) exemplifies an apparent preference for Tm-5°C. Note, although all the above amplifications were performed simultaneously under identical conditions and using gradient PCR machine, no quantitative estimate of the amplification efficiencies can be made. However, relative differences due to the change of the annealing temperature could and have been detected. (B) A gel electrophoresis exemlifying RACE-PCR amplification of Agelena orientalis cDNA using hybrid primers (left to right) M31F, M33F, M35F, M37F, M39F, M41F, M43F, M45F, M47F, M49F, M51F, M31F (identical to the leftmost PCR product). Nearly all primers (except for M37F and M47F) yielded more than one group of products. Amplification conditions were optimised for each hybrid primer, as described above (panel A) and the oligo-dT primer with the matching Tm (see Table 1). (C) High throughput positive clone selection by direct PCR amplification from bacterial colonies. The lower band (just above 100 bp marker) corresponds to amplified plasmid fragments without inserts. The upper bands are the amplified fragments with inserts. Plasmid specific primers PBS-F and PGM-2R were used (see Table 1 for sequences).