摘要
采用cDNA合成技术制备文昌鱼头部cDNA基因文库 .以mRNA为模板 ,用NotIprimer Adaptor为引物 ,在反转录酶的作用下 ,合成第一链cDNA ,进而合成第二链cDNA .含有EcoRI和NotI粘性末端的双链cDNA在T4DNA连接酶作用下与λgt1 1EcoRI/NotI臂相连 ,包装蛋白包装 ,得到噬菌体颗粒 ,即原始的文昌鱼头部cD NA文库 .经X Gal/IPTG板测定 ,该文库滴度为 7.8× 1 0 5 pfu/cm3 ,重组率为 70 % .
The cDNA library in the head region of amphioxus, Branchiostoma belcheri was constructed using cDNA synthesis technique. The first strand cDNA was driven by AMV reverse transcriptase and NotI primer-Adaptor in the presence of mRNA template, followed directly by second strand replacement synthesis using RNAse H and DNA polymerase I. The double stranded cDNA with EcoRI and NotI cohensive ends was ligated to EcoRI/NotI λgt11 arms. The ligated DNA was added to a packaging extract and the packaged phage referred to as original cDNA library was obtained.Titration of packaged phage on LB plate containing X Gal/IPTG indicated that the titre of the cDNA library was 7.8×10 5 pfu/cm 3 and recombinant phage accounted for 70%. The resultant cDNA library will be used for the screening of target genes.
出处
《台湾海峡》
CAS
CSCD
2003年第2期131-135,共5页
Journal of Oceanography In Taiwan Strait
基金
国家自然科学基金资助项目 ( 39870 12 0 )
