02琼脂糖凝胶电泳解剖.ppt

2005-4-12 2003级生物科学专业 2005-2006(上)分子生物学实验 Gel electrophoresis 二、实验原理(Experimental Principle) Electrophoresis is a technique used to separate and sometimes purify proteins and nucleic acids, which differ in size, charge or conformation from each other. Electrophoresis has become the most widely used techniques in biochemistry and molecular biology. Agarose is a polysaccharide extracted from seaweed. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. Agarose is typically used at concentrations of 0.5% to 2%. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Gelview is a fluorescent dye. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of Gelview. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. 三、试剂与器材（Reagents and apparatus） 四、实验步骤(Experimental Procedures) Ⅰ. Preparation of the gel （凝胶的制备） 1.制备1％琼脂糖凝胶。称取0.5g琼脂糖，放人锥形瓶中，加入50mL的 0.5×TBE 缓冲液，放入微波炉加热至完全溶化，则为1％琼脂糖凝胶液。（由于蒸发作用，溶解前在容量瓶上作一个记号，溶解后用三蒸水补足） 2.制胶器的安装 ①取多功能制胶器，洗净，晾干； ②将多功能制胶器放置于一水平位置，选择12×6cm制胶架，然后选择1.5mm 18teeth的梳子（最大加样量25μl）； ③将所选择规格的梳子插入制胶架的定位槽中。 3. 将熔化的琼脂糖凝胶液转入Gelview专用的三角瓶中，然后加入Gelview 5μl。 4. 将冷到60℃左右的琼脂糖凝胶液，缓缓倒入所选择的制胶槽内，直至有机玻璃板上形成一层均匀的胶面(注意不要形成气泡)。 5. 待胶凝固后（30-60min），轻轻拔掉梳子，将凝胶盘从制胶槽中取出，放入电泳槽内。 6. 加入电泳缓冲液(0.5×)至电泳槽中。 Ⅱ. Loading DNA samples (加样) 用移液枪缓慢将DNA样品垂直加入加样孔直至开口下方。 1.DNA samples ： 10μl 2. Loading buffer (6X)：2μl 3. DNA markers ：6μl Ⅲ.Gel（电泳） 1. 接通电泳槽与电泳仪的电源(注意正负极，DNA片段从负极向正极移动)。保持电压 60－80 V。 2. 当溴酚蓝染料移动到距凝胶前沿1-2cm处，停止电泳。 Ⅳ. Gel Interpretation （凝胶图像解释） The uncut DNA lane may have several bands in it. This occurs because the mobility of plasmid DNA in an agarose gel