Staged Construction Of Osteosacoma Genomic DNA Library

Objectives:①To supply experiment platform of study on the pathogenesis, multidrug resistance, gene diagnosis and treatment of Osteosacoma on gene level②To prepare DNA fragment compatible with library construction and EMBL4 vector for staged construction Osteosacoma genomic DNA library ,and on which to base the complete later stage construction.Methods:①Employ phenol to extrct high molecula weight DNA from Osteosacoma tissue: by means of clearage buffer which contains EDTA,SDS and RNA enzyme with noneDNA to clearage the cells,after dealed with by prolease K , use Tris saturated phenol and chloroform to extract DNA repeatedly and precipitate DNA;②Choose Sau3AI as the restriction endonuclease to digest DNA, according to the result of small-scale reaction ,determine the optimal enzyme concentration,then perform larg-scale partial digestion of genomic DNA ;③Choose common agarose method to extract desired DNA fragments:Firstly perform electrophoresis in a 0.5% agarose gel ,cut the gel where desired fragments gather,then use phenol, chloroform to extract them,and ammonium acetate , dehydrated alcohol to precipitate them,analyze the quality by electrophoresis;④Preparation of EMBL4 vector: Digest uncut vector by BamH I and EcoR I to lower nonrecombinant backround,then remove oligonucleotide, dephosphorylate.Results:①The DNA extrcted from Osteosacoma tissue by phenol method,whose molecula weight >80kb,fits the requirement of library constrction that the molecula weight of DNA should be at least as 3-5 times as the one of the library's final average inserted fragments,analyze by ultraviolet ray absorptiometer :A260\ A280=1.83,DNA purity is high;②At37℃, employ Sau3AI to digest Osteosacoma genomic DNA ,whose concentration is 0.0033u/...