DNA凝胶电泳是用来检测和分离DNA分子的一种技术。施加一个电场到由琼脂糖构成的凝胶基质上，凝胶中的带电粒子将根据其大小进行移动和分离。DNA骨架上带负电的磷酸集团会使得DNA片段移向阳极 – 它是带正电荷的电极。
本视频解释了DNA片段在琼脂糖凝胶上分离的原理， 并提供了一个常用的逐步操作过程，包括如何准备琼脂糖凝胶，上样DNA样品，DNA电泳跑胶，观察DNA片段，以及实验完成后正确地处理凝胶和电泳缓冲液。
综述
DNA凝胶电泳是根据DNA分子大小来分离和识别DNA片段的技术。将不同大小的DNA片段上样到由琼脂糖制成的多孔凝胶上 – 琼脂糖是一种从红藻中发现的碳水化合物。施加电场后，由于DNA上有携带负电荷的磷酸集团，片段会在凝胶中移动。较小的DNA片段在凝胶中的移动会比较大的片段要相对容易，较大的DNA片段在凝胶中的移动会更难。当电泳结束时，可比较您的DNA样品所在的位置和一系列已知大小的DNA片段的位置，这些已知大小的片段称为DNA分子量标准样。您的目的DNA片段可以根据它的大小来确认，而它的大小则是通过比较的实验样品和分子量标准样之间的相对位置来判断。
溶解琼脂糖和制备凝胶
琼脂糖凝胶是根据质量体积比来制备的。所以1克琼脂糖和100毫升电泳缓冲液可以制备1%的琼脂糖凝胶。低浓度的琼脂糖凝胶分离较大的片段效果较好，而高浓度的凝胶则更容易区分较小的片段。要制备琼脂糖凝胶，首先称取适量的琼脂糖到锥形瓶当中。然后加入适量的电泳缓冲液，注意体积不要超过锥形瓶的三分之一。旋转锥形瓶混匀。将锥形瓶置于微波炉中用最大火力加热来溶解琼脂糖和缓冲液混合物。每隔30秒钟，取出锥形瓶旋转混匀。重复这一过程直至琼脂糖完全溶解。
下一步则是加入溴化乙锭至浓度为0.5毫克每毫升。溴化乙锭是一种芳香族化合物，能够嵌入DNA的相邻的碱基之间，然后在紫外线照耀下，使DNA释放出橘黄色的荧光。必须注意的是溴化乙锭是一种致癌物质，所以在操作含有该种化合物的琼脂糖凝胶时，应当时刻戴上手套。
为防止凝胶槽高温变形，先将溶解好的琼脂糖凝胶溶液至于65度水浴中冷却。冷却过程中，准备凝胶模具，可将凝胶槽置于制胶板中。也可使用胶带密封凝胶槽两端的开口，从而形成一个模具。将梳子放到胶中形成上样孔，DNA样品将由此加入。 要注意选择合适的梳子来制成适合您DNA样品量的上样孔。将溶解的琼脂糖凝胶倒入模具当中，然后在室温下凝固。当琼脂糖凝胶凝固后，取下梳子。如果短时间内不使用凝胶，可以用塑料薄膜将其包裹然后置于4度保存直至使用。如果要立即使用凝胶，则将其置于电泳槽当中。
准备和跑胶
在开始电泳之前，先将上样缓冲液加入需要分离的DNA样品中。上样缓冲液通常是6倍浓度，它使得DNA样品在加入上样孔的时候肉眼可见。而且在电泳过程当中，可以指示DNA样品在凝胶上移动的距离。
设定电源到指定的电压。向电泳槽中加入足够的电泳缓冲液直至覆盖凝胶表面。记得使用和制备凝胶时同样的电泳缓冲液。
观察分离的DNA片段
电泳结束后，关闭电源，移去电泳槽盖。从电泳槽中取出凝胶，倒干表面的多余的电泳缓冲液。将胶槽置于纸巾上吸走剩余的电泳缓冲液。
为观察DNA片段，先将凝胶从胶槽中取出，用紫外灯照射。DNA片段应当显现为橘黄色荧光带，给凝胶拍照。
实验结束之后，根据所在院校规定正确处理凝胶和电泳缓冲液。记住在处理凝胶和电泳缓冲液的时候，要戴手套以防止接触到溴化乙锭。
应用和改动
现在您已经知道如何进行DNA琼脂糖凝胶电泳。下面让我们来看一下这个非常有用的方法的一些下游应用及其改动。
这是一个DNA琼脂糖凝胶电泳结果，用于分离PCR产物。可看到上样的DNA片段电泳之后被清晰地分离。DNA分子量标准样应当分离到能足够帮助判断样品带的大小。在这个例子中，使用1.5%的琼脂糖凝胶分离分别为765bp, 880bp, 1022bp的DNA片段，并用了2倍-对数梯度DNA分子量标准样。
DNA凝胶电泳除了用于确认您目的DNA片段的存在，还可是凝胶纯化中的一个步骤。 通常是使用刀片切下并收集含有目的DNA片段的凝胶，这样就可回收凝胶中的DNA样品。
琼脂糖凝胶电泳的下一步还可以是印迹转移，将DNA或者RNA转移到硝酸纤维素膜上，然后使用放射性的探针去检测电泳分离样品中特定的DNA或者RNA的序列。
对于分离大于15kb到20kb的大分子量DNA片段，如基因组，标准的DNA琼脂糖凝胶电泳并不是最佳手段。要分离大分子量DNA样品，可以采用脉冲场凝胶电泳。这是一种可以将凝胶内的电场方向改变或者以脉冲的方式释放的技术。这种技术涉及到一种特殊的凝胶电泳装置，它在凝胶周围的不同方向上都有成对的电极。该技术可用来检测不同种群在基因组大小上的差异，例如这里从不同的湖泊环境中的不同微生物群落中提取出的DNA混合物。
结语
您刚观看的是对DNA琼脂糖凝胶电泳的介绍。我们演示了该方法的基本原理，如何制备琼脂糖凝胶，如何进行电泳跑胶，如何分析结果，以及一些琼脂糖凝胶电泳的常见应用。感谢观看并预祝您电泳跑胶成功。
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