1.华中科技大学基础医学国家级实验教学示范中心;2.华中科技大学同济基础医学院药理学系
] 国家自然科学基金委员会面上项目(82273911)：骨-脑轴介导的lipocalin 2/MC4R通路异常在围绝经期抑郁症中的机制研究
1.National Demonstration Center for Experimental Basic Medical Education （Huazhong University of Science and Technology）;2.Department of Pharmacology，Tongji Medical College，Huazhong University of Science and Technology
目的 为模式小鼠基因型批量鉴定建立简便、快捷、经济的DNA提取方法。方法 以鼠尾组织为实验材料，采用碱煮简化法、碱煮常规法、蛋白酶K裂解法和DNA提取试剂盒四种方法提取模式小鼠DNA，测定 DNA 模板纯度、浓度，评价凝胶电泳图的优劣，并比较四种方法所耗时长及实验成本。结果 蛋白酶K裂解法提取的DNA浓度最高，碱煮简化法和常规法次之；试剂盒处理所得的DNA纯度较高，碱煮简化法次之。四种方法处理所得的DNA模板PCR扩增、凝胶电泳后均可得到清晰的DNA目的条带，且在 -20 ℃ 保存一个月后也可用于基因鉴定。此外，碱煮简化法成本最低且耗时最少。结论 模式小鼠批量鉴定基因型时，碱煮简化法是目前最快捷、最经济的DNA模板提取方法。
Objective To establish a simple，fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice. Methods Four methods，i.e.alkali boiling routine method, alkali boiling simplified method, protease K lysis and DNA extraction kit methods were used to extract the total DNA from transgenic mice tails．The purity and concentrationof DNA obtained by the four methods were measured, the advantages and disadvantages of gel electrophoresis were evaluated, and the time-consuming and experimental costs of the four methods were compared．Results The DNA concentration extracted by protease K cleavage method is the highest, followed by alkaline boiling simplified method and conventional method; The DNA purity obtained from the reagent kit treatment is relatively high, followed by the alkaline boiling simplification method. The DNA templates obtained by four methods can be amplified by PCR and Gel electrophoresis to obtain clear DNA target bands, and can also be used for gene identification after being stored at -20 ℃ for one month. In addition, the simplified alkaline boiling method has the lowest cost and the least time consumption. Conclusion The alkaline boiling simplification method is currently the fastest and most economical DNA template extraction method for batch genotyping of model mice.
