cDNA Library Construction of Testis and Ovary ofScylla serrata with SMART Technique

To clone the testis and ovary specific expression genes of Scylla serrata,we constructed two full-length cDNA libraries using the SMART cDNA library construction kit(Clontech).Total RNA were prepared from testes and ovaries using RDP,a reagent modified from Chomczynski and Sacchi(1987)for the simultaneous isolation of RNA,DNA,and protein.Oligotex(QIAGEN)was used to separate the mRNA of testes and ovaries from total RNA.Theanchor first-strand cDNAcontaining asymmetricalSfiⅠrestriction enzyme sites(AB) was synthesized by transcription of mRNA with the SMART technique.The LDPCR was performed using a modified oligo(dT)primer and an anchor primer as the primer set, and anchor first-strand cDNA as the template to enrich the cDNA population for full-length sequences.After digestion withSfiⅠand size fractionation using CHROMA SPIN-400~(TM) Columns,SMART cDNA was ligated into theSfiⅠ-digested λTriplEx2~(TM) Vector.The ligation mixture was packaged into lambda capsids using the Gigapack Ⅲ Gold packaging extract to generate full-length testis and ovary cDNA libraries ofScylla serrata.The titer of un-amplified cDNA libraryies was 1.45×10~6 for testis library and 1.75×10~6 for the ovary library.The cDNA inserts size were between 400 bp～3 kb.These results indicate that the quality of these cDNA libraries is good enough for further cloning of the testis and ovary specific expression genes.