A Rapid and Simple Method of cDNA Library Construction

AIM:To establish a method of cDNA library construction based on long-distance RT-PCR.METHODS:Total RNA from the regenerated hepatic tissues were isolated,and mRNA were separated and purified through the magnetic substance combined with Oligo(dT).The poly-deoxythymidines and poly-deoxycytidines were introduced into the 5′ terminal and 3′terminal of first cDNA strand with the help of M-MLV reverse transcriptase and terminal transferase,respectively.Then double strand cDNA with the restriction site of BamH Ⅰ and Hind Ⅲ were synthesized through LD-PCR(Long-distance-PCR) and ligated into the pUC19 vector,recombinant vectors were transformed into E.coli DH5α and amplified,qualities of the cDNA library were analyzed.RESULTS:The average capacities of the cDNA library is about 5.84×10~(5)clones per μg ds-cDNA with recombant rate of 96.7%.Among the 25 detected randomly selected cDNA clones,none repeated inserted cDNA fragment were founded.CONCLUTION:cDNA library constructed with this method is suitable for functional genomic analyzing.