Construction And Identification Of A Full-length CDNA Library For Xingkai Lake Sika Deer Liver

The total RNA was separated from Xingkai lake Sika deer liver tissues using TRIZOL method.SMART technique and COSⅢ/3' primer were used for first-strand eDNA synthesis. LD PCRs werethen used to analyse the double-strand cDNAs that were then digested by SfiI and fractionated byCHROMA SPIN-400 columns. The longer 400bp cDNAs were collected and linked toλTripIEx2 vector.Thenλphage packaging reaction and library amplification were performed. The qualities of original andamplified cDNA libraries were strictly checked by conventional titer determination. Sixteen plaqueswere randomly picked and tested using PCR with universal primers derived from the sequence flankingthe vector. The titer of primary cDNA library was 7.9×10~5pfu/ml, the titer of amplified library was1.06×10~9pfu/ml and the rate of recombinant was above 88%. The insert size ranged from 0.3～3.0kb.Therefore, a cDNA library of Xingkai lake Sika deer liver tissues has been successfully constructed.The full-length eDNA library of Xingkal lake Sika deer liver tissues has been constructedsuccessfully by SMART technology, which was essential for screening and cloning of new genes.Furthermore, Accomplishment of this experiment is an initial key for building the resourcefulinformation databank of the Xingkai lake Sika deer.