用λgt11载体构建人脑胶质瘤基因文库

Construction of the Human Glioma cDNA Library Using λgt11 Vector

自人脑胶质瘤手术标本中提取mRNA,逆转录合成cDNA,在T_4DNA 连接酶作用下,与一头是平末端,另一头是EcoR I 粘性末端、内含Not I 切点的插头相连,经T_4多聚核苷酸激酶磷酸化后,再与脱磷酸处理过的λgt11 臂连接,形成重组λgt11DNA。体外包装,构建了一个库容量含1.02×10~6重组子的人脑胶质瘤cDNA 基因文库。克隆效率为1.08×10~7pfu/μg cDNA。重组百分比为96.2%。

RNA were purified from two specimens of human glioma obtained from operation.The double—stranded cDNA molecules were synthesized by reverse transcription.The eDNA molecules were lig-ated to adaptors containing an internal Not I site,a blunt end and a preformed EcoR I cohesive endat the other end.After the adapted cDNA molecules had een kinased,it was able to be ligated to dephosphorylated λ gtll vector arms.This resulted in recombinant λgtll DNA molecules.The hymanglioma cDNA library was constructed by in vitro packaging.The library contained 1.02×10~5 reco-mbinants.Cloning efficiency was 1.08×10~7 pfu/μg cDNA.Percentage of recombinants was 96.2%.