质粒纯化是将质粒DNA与基因组DNA、蛋白质、核糖体以及细菌细胞壁分离开的一种技术。质粒是小的环状双链DNA，用作特定DNA分子的载体。当质粒通过转化技术导入宿主细胞后，将被复制产生大量拷贝的DNA片段用于研究。
本短片讲述了常用的质粒纯化的逐步过程。质粒纯化包括三个基本步骤：细菌的培养，细菌的收集和裂解，质粒DNA的纯化。视频还解释了操作过程每一步中质粒在哪里会被发现，以及如何用分光光度计和电泳对质粒进行定量和纯度的分析。目前有多种质粒纯化的方法，可以根据所需质粒的产量，质粒的拷贝数目和细菌培养的体积来进行选择。
质粒纯化
质粒是独立于染色体的环状DNA分子。在分子生物学中，他们被用作运送分子，或称载体，用于承载特定的DNA片段。利用细菌来复制质粒，可以大规模生产目的DNA。这个被研究人员用来从细菌中获得质粒的方法称为质粒纯化，将在本短片中得到讲解。
质粒纯化步骤中的关键因素
质粒纯化当然是指纯化质粒，但这具体是什么意思呢？要纯化质粒就是将质粒从细菌的染色体、蛋白质、细菌胞膜和细菌核糖体中分离出来。
尽管纯化质粒的试剂盒有很多种，但它们的基本原理都是一样的。首先，挑取细菌菌落在含正确抗生素的合适培养液中生长。由于质粒有编码抗生素抗性基因，只有那些含有该种质粒的细菌才能在这种培养液中生长。然后收获细菌，在高pH条件下进行裂解。接下来中和pH，加入盐，最后离心去除碎片和基因组DNA。
质粒纯化的逐步步骤
在操作中，应当穿戴合适的实验服，一次性手套和防护眼镜。
将携带目的质粒的细菌加入到含有合适抗生素的培养液中，在37摄氏度摇床上培养过夜。
第二天，离心收集细菌沉淀，弃去上清。然后用重悬缓冲液重悬细菌沉淀。
重悬之后，细菌被转移到一个较小的离心管中并加入裂解缓冲液。裂解缓冲液具有较高的pH值并含有去污剂，如十二烷基硫酸钠。它能够破坏细菌的细胞膜从而裂解细菌。刚加入裂解缓冲液时溶液会浑浊。混匀后将变得澄清。不要震荡混合液，否则可能会造成基因组DNA断裂，从而污染质粒DNA纯化。
然后加入中和缓冲液来中和碱性环境并降低pH。轻轻混匀，这样基因组DNA和与之结合的蛋白质会沉淀下来，而质粒DNA则会留在溶液中。这里同样也要避免震荡，防止基因组DNA污染质粒DNA。
离心混合物，使基因组DNA和蛋白质沉淀在底部。上清中则包含质粒DNA和可溶性蛋白。
将上清轻轻倒入或用移液枪转移到硅胶柱中，弃去沉淀。
接下来，用高盐缓冲液过柱，DNA仍将吸附在硅胶柱上。用高盐缓冲液反复洗涤硅胶柱，将去除掉核酸内切酶，核糖核酸，蛋白质，以及小分子量的杂质。过柱的高盐缓冲液应当丢弃。最后一步洗涤后，要确认硅胶柱完全干燥，没有任何缓冲液残留。此时，质粒DNA仍然结合在硅胶柱上。
使用无菌水或洗脱缓冲液来洗脱质粒DNA。得到的DNA可以被紧接着应用到很多方面。
纯化完成后，有许多方法可用来检验质粒的纯度。分光光度计可以通过比较在不同波长条件下的吸光度来测定质粒DNA的浓度。琼脂糖凝胶电泳也可用来分析质粒DNA的大小是否正确或者是否有杂质。这一步用来确认质粒没有被基因组DNA污染并且没有在细菌内被修饰。
应用及改动
对于不同的质粒大小、拷贝数、和培养体积，可相应改动该质粒纯化过程，包括使用不同的结合，清洗和洗脱设备。其中最主要的区分标准是质粒产量，它将质粒制备划分为微量提取，中量提取，大量提取和超量提取。
关于下游的应用，质粒纯化后通常会被用于一种叫做转染的技术。它是将质粒DNA转入到真核细胞当中。多数时候，转染的目的是利用报告蛋白来观察细胞或组织的结构，例如您这里看到的这些被标记的神经元。
有时，分别纯化的多个质粒会被转入到同一细菌当中，这些质粒编码几种酶，从而在细菌中再造形成一个完整的生物合成途径。最终结果是合成一种复杂的化合物，例如您这里看到的利用细胞生产的抗生素。
纯化的质粒也可能用来重新转化细菌，用于大量生产该质粒编码的蛋白质。这里您看到的是将细菌细胞破碎和裂解后，通过亲和层析来分离目的蛋白。然后结晶纯化的蛋白来进行结构分析。
结语
您刚观看的是JoVE对质粒纯化的介绍。本短片中，我们讨论了该方法的基本原理，实验的逐步介绍，以及质粒制备的多种应用。我们一如既往地感谢您的观看。
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