Construction of a cDNA library from microdissected guinea pig organ of Corti

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.