The construction of the nanobody library followed the previously described protocol[23]. Briefly, the DNA library of nanobodies was constructed by two-step overlap-extension PCR (OE-PCR). A set of ten primers[23] (Tsingke Biotechnology Co., Ltd.) were dissolved and mixed to prepare three mixed pools, ‘mix short’, ‘mix medium’, and ‘mix long’, differed in CDR3 region of variable length of 7, 11, or 15 randomized residues respectively. The full-length nanobody DNA product from each pool was mixed in a 1:2:1 molar ratio of short/medium/long CDR3 regions, referred to as the nanobody DNA library pool hereafter, recapitulating the length distribution frequencies observed in camelid VHH domains.|||In brief, RT was performed using the ExScript RT reagent kit (Takara) in a final volume of 20 mL containing 1 μg total RNA, 4 mL 5X ExScript buffer, 1 mL deoxynucleotide triphosphate (dNTP, 10 μM) mixture, 1 mL oligo(dT) primer, 0.5 mL ExScript RTase, 0.5 mL RNase inhibitor and RNase-free water. PCR was conducted according to the instructions of TsingKe Golden mix under the following conditions: pre-DNA denaturation at 98°C for 2 minutes; DNA denaturation at 98°C for 10 seconds; annealing for 30 seconds at 60°C; elongation was carried out at 72°C for 10 seconds; the total cycle number was 30. All experiments were performed in triplicate.
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