Isolation of antigen specific nanobodies from camelid blood

摘要

ISOLATION OF ANTIGEN SPECIFIC NANOBODIES FROM CAMELID BLOOD Method of isolation of antigen-specific nanobodies from camelid blood comprising the following steps : a) following immunization protocol of suitable breed of camelid as desired, b) separation of peripheral blood lymphocytes, c) Isolation of RNA from Peripheral Blood Lymphocyte, d) synthesis of first-strand c-dna, e) First PCR reaction involving using suitable reagents and setting temperature 95ºC for 5 minutes, 95ºC for 45 Sec, 57ºC for 45 sec, 72ºC for 45 sec, 72ºC for 10 minutes, 4ºC for storing and the PCR was ran for 35 cycles, f) Gel eluting first pcr product in the manner such as herein described, g) Second PCR reaction, h) Purification of product digest by PstI and NotI, i) plasmid isolation (PHEN4 vector) – maxi preparation in the manner such as herein described, j) harvesting cell, k) plasmid purification, l) digestion of PHEN4 vector, by PstI and NotI in the manner such as herein described, m) gel eluting digested phen4 product, n) ligation reaction, o) competent cell preparation of TG1, p) transformation involving taking 200 ult transformed cells and putting on LB agar plate containing ampicillin, q) undertaking colony screening, r) undertaking colony PCR and finally s) preparation of phagemids.