摘要
以大麦为材料,用硫氰酸胍-热酚-氯化锂-蛋白酶K法抽提萌发种子的总RNA,经过Oligo(dT)-纤维素柱层析,分离出总mRNA。经MuMLV逆转录酶合成cDNA第一条链,再用E. coliDNA聚合酶Ⅰ-RNase H共同作用,合成第二条链。经DNA聚合酶Ⅰ大片段修平末端后,与经过SmaⅠ完全酶切并用碱性磷酸酶处理了的pUC19载体连接,转化受体菌JM107,挑出白色重组子,用小麦全长的α-淀粉酶基因为探针经过菌落原位杂交筛选,获得一个含有α-淀粉酶基因片段的阳性克隆,插入片段长度约0.8kb。
Total RNA of germinated barley seed was extracted using guanidin thiocynate/hotphenol mothod followed by LiCl precipitation and proteinase K digestion, and total polyA^+ RNA was obtained by oligo (dT)-cellulose chromatography. First strand of cDNAwas syntherized using MuMLV-reverse transcriptase and the second strand synthersizedwith the joint action of RNase H and DNA polymerase I. Double stranded cDNA wasflushed with Kleno fragment of DNA polymerase I to get perfect blunt ends, and ligatedwith SmaI digested pUC 19 pretreated with alkaline phosphatase. E. coli JM107 wastransformed with the ligation mixture and about 600 recombinants were obtanied fromthe first batch of transformation. One clone containing 0.8kb α-amylase cDNA wasisolated through in situ hybrization.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
1990年第3期315-318,共4页
Journal of Huazhong Agricultural University
基金
农业部七五重点项目农02-01一03资助
