Objective The purity of DNA for microinjection is a crucial factor for preparing transgenic animals.The aim of this study is to develop an improved DNA purifying method for preparation of DNA fragments in high purity,feasible for routine transgenic animal research work.Methods DNA fragments containing lumbrokinase gene were extracted and purified by phenol-chloroform repeated extraction method and routine gel extraction kit.The purified DNA was microinjected into pro-nucleus of mice fertilized eggs and the injected living eggs were transplanted into the oviducts of pseudo-pregnant mice.The offspring after transplantation were identified by Southern blot.The two purifying methods were compared according to the results of transgenic experiments.Results Transgenic mice could be obtained with DNA purified by both two methods.There was no notable difference in DNA purity and viability of injected eggs,but higher birth rate of transplanted eggs and transgenic positive rate were obtained by the extraction method than those obtained by the kit method.Conclusion Our findings indicate that the extraction method can be used in purifying DNA for microinjection,replacing the kit method,with advantages such as lower cost,simplified experimental procedure,and improved transgenic positive rate in preparation of transgenic animals.