Wilcox E R, Fex J
Laboratory of Molecular Biology, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892.
Hear Res. 1992 Sep;62(1):124-6. doi: 10.1016/0378-5955(92)90208-5.
Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.
从显微切割的豚鼠柯蒂氏器中分离出多聚腺苷酸（Poly (A)）RNA，并使用核糖核酸酶H - 鼠白血病病毒逆转录酶将其转化为互补脱氧核糖核酸（cDNA）。经过大小分级分离后，将cDNA定向连接到载体pSPORT 1中，并通过电穿孔将质粒转化到DH10B大肠杆菌中。发现该文库有3.35×10⁶个独立菌落，其中10%的菌落没有插入片段。在随机检查33个菌落以确定是否有插入片段后，平均插入片段大小为1218个碱基对，范围从3300个碱基对到400个碱基对。用β - 肌动蛋白寡核苷酸探针筛选该文库，1.4%的菌落含有与探针杂交的插入片段。
