一种从非变性聚丙烯凝胶中回收小片段DNA的方法

摘要

利用高分辨率的非变性聚丙烯酰胺凝胶电泳是分离、纯化特异性小片段DNA的首选方法,也是开展后续多种分子生物学试验的前提.本试验比较了改良煮沸法(液氮研磨+煮沸+低温浓缩)、煮沸法及直取法回收小片段DNA,以回收样品模板进行第二次PCR扩增,结果表明:改良方法回收的DNA的纯度高,特异性好,回收率与经典煮沸法相当.在保证回收产物的特异性时,用低温浓缩法替代乙醇/醋酸钠共沉淀也具有可行性及一定的创新性.%The polyacrylamide gel electrophoresis with high resolution is a top choice for isolating and purifying specific short DNA fragments, and also serves as a prerequisite for a variety of subsequent molecular biology experiments. In this study, three methods, i.e., modified boiling method(grinding with liquid nitrogen + boiling + concentrating at low temperature), traditional boiling method(in which DNA is precipitated with absolute ethanol and 3 mol/L sodium acetate together), and direct recovery method were used to extract short DNA fragments. The short DNA fragments were used as the template for a second PCR reaction. The results showed that recovered DNA fragments by the modified boiling method had higher purity and finer specificity, and the recovery rate was nearly the same as the traditional boiling method. Replacing the method of precipitation with ethanol and sodium acetate by method of concentrating at low temperature is feasible and novel.