Development of method for preparation of high molecular weight DNA from Citrus grandis (L.) Osbeck 'Guanximiyou'.

High molecular weight nuclear DNA(HMW-DNA)from leaves of Guanximiyou [Citrus grandis (L.) Osbeck ‘Guanximiyou’] was prepared by two methods(Zhang’s and Peterson’s method)and the extracting effects of the two preparing methods were compared.The results showed that Zhang’s method was more suitable for preparation of HMW-DNA in Guanximiyou.The cell walls were broken down by gentle physical homogenization to keep integrity of nuclei under suitable osmolarity.Debris of cell walls and most of polysaccharides were removed by filtering with miracloth and centrifugation under slow speed.The isolated nuclei were proved to be intact,pure,and high-quality by observation under microscope and were embedded with low-melting-point agarose to preserve the isolated nuclei in plugs for keeping stability of nuclei.The optimum digestion time of plugs was 48 hours,the pulse field gel electrophoresis (PFGE)results showed that the isolated HMW-DNAs were more than 1 Mb in size and were digested completely or partially by restriction enzymes,therefore,Zhang’s method was suitable for construction of BAC library in Guanximiyou.