Normalized cDNA Library Construction of Ovary of Scylla serrata

Total RNA was isolated from ovary of Scylla serrata.Oligotex(QIAGEN) was used to isolate the mRNA from the total RNA.The first strand cDNA was synthesized by transcription of mRNA with the SMART technique.The LD-PCR was performed using a modified SMART primer as the primer set,and first-strand cDNA as the template to synthesize double strand cDNA.After treatment with DSN,the normalized cDNAs were digested with Sfi I enzyme and purified with gel purification kit to remove the small PCR products(500bp).These normalized cDNAs were ligated into the Sfi I-digested pDNR-LIB Vector.E.coli(TOP10) were transformed with the ligation mixture to generate a normalized cDNA library.The titer of unamplified cDNA libraries was 4.7×104cfu/ml.The titer of amplified cDNA libraries was above 108cfu/ml.The cDNA inserts sizes ranged between 0.5-2.5kb.These results indicate that the normalized cDNA library is suitable quality to be used for further cloning and analysis of genes related to gonad development in Scylla serrata.