Construction of cDNA Library from the Marrow of Sika Deer

Objective To construct a cDNA library from the marrow of Sika Deer. Methods Total RNA was extracted from the marrow of Sika Deer by using TRIzol Reagent.mRNA was purified with mRNA Purification Kit.Double-stranded cDNA were synthesized through reverse transcription.Blunted the cDNA termini,ligated the EcoR I Adapters and phosphorylated the EcoR I ends.Digested cDNA with Xho I,removed short fragments by column chromatography fraction.Connected vector pBluescript Ⅱ SK(+) and electrotransformated to competent cell DH10B,then,the number of clones,recombinant rate and the length of insert fragments were checked. Results The capacity of library was 3.5×106 with a recombinant rate of 94%,and the length of insert fragments ranged from 0.5 to 4.5kb,the average length was about 1.5kb. Conclusion The constructed library can meet the requirement for screening of known and unknown genes from sika deer,and provide a basis for further study of gene structure and functions.