Synthetic Phage Display Library Construction

Cascade Temperature Assembly

An equimolar amount of each segment (100 nM) was added to a total reaction of 20 ul, containing 20 mM Tris–HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 0.1 mg/ml bovine serum albumin (BSA), 0.1% (v/v) Triton X-100, 2 mM MgSO4, 0.5 mM dNTP, and 1.25 U Pfu DNA polymerase (Fermentas, Vilnius, Lithuania). The mixture was heated at 95 oC for 2 min 30 s, then at 90 oC for 2 min 30 s, and then slowly cooled down to 85, 70, 60, 50, 40, 30, and 25 oC, for 10 min at each temperature. The temperature ramp was set at 0.1 oC/s, starting from 95 oC.

Amplification of the assembled full-length gene

Add 6 ul first reaction  to 20 ul PCR mix and amplified with 10-fold concentration of the outermost primers. The amplification profile used was 95 oC for 1 min 30 s; 30 cycles of 95 oC for 30 s, 61 oC for 30 s, and 72 oC for 1 min; and last, 72 oC for 5 min. After the amplification, the assembled VH gene was subjected to gel electrophoresis (1.2% (w/v) agarose gel) and purified using a gel extraction kit according to the manufacturer’s instructions