Test of CS length, reaction temperature and incubation time.

<p>(<b>A</b>) To analyze the influence of the catching sequence length on cloning efficiency, the T019 GC3F PCR product was cloned into pICH31477 (23 nucleotide CS) and pICH31480 (52 nucleotide CS). Cloning was performed using an incubation at 15°C for 0, 5, 10, 20 and 30 minutes (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence) (<b>B</b>). (<b>C</b> and <b>E</b>) Eight randomly chosen clones from reactions with the incubation times that yielded the most clones were analyzed by colony PCR using vector primers. The PCR products were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow. (<b>D</b>) To determine the optimal incubation temperature and time, the T019 GC3F PCR product was cloned into pICH31480 (52 nucleotide CS) using incubation temperature of 4°C, 15°C and 25°C for 0, 5, 10, 20, 30 and 60 minutes.