Construction Of A Full-length CDNA Library Of Antler Mesenchymal Cell

This experiment obtained the mesenchyme from the antler by the histology dissection. Then we digested the material with the trypsin and cultured the antler mesenchymal cells. Subsequently, we taked a series of experiments to construct the cDNA library, such as, extracting the total RNA form antler mesenchymal cells with TRIZOL, reverse transcription to get single stranded cDNA, synthesis double strand cDNA with LD-PCR method; purification PCR product after the proteinase K hydrolysis, cutting with the SfiI enzyme; classification sepearation the digested product, retrieving the cDNA component above 500bp and connection withλTriplEx2 carrier; The connection product after in vitro protein packing, producing the primary library; After identificating the library titer, taking the library to amplification. Randomly choosing a number of macrophages plaque, with the universal primers of vector cloning sites for PCR amplification to detect the quality of the cDNA library. The initial determination of the library titer to 3.0×105 pfu / ml, after amplified, the titer was 6.0×108 pfu / ml. Insert fragments'length were more than 500 bp and many of them were more than the 1000 bp fragments. Moreover, using the additional sequences methods to make the full length mRNA reverse transcription, the product of reverse transcription were cDNA, which were intact and fully met with the requirements of cDNA library. That is, we successfully constructed a antler mesenchymal cells cDNA library.This experimented had constructed the antler mesenchymal cells cDNA library successfully. It had the solid foundation for the antler fast growth research through antler mesenchymal genes, would also help the antler regeneration mechanism study.