热激法转化细菌的介绍
细菌转化是一种应用广泛的方法，它将外源DNA导入细菌，用以扩增或者克隆DNA。能够摄入外源DNA的细胞被称为感受态细胞。尽管自然界中许多类型的细菌可发生转化，科学家们已经找到方法来人工诱导和增强细菌细胞的感受性。本 短片中，我们将讲述这些方法中的一个：热激转化。
热激转化的基本原理
在介绍热激转化技术之前，让我们先讨论一下在细菌转化中最常用的DNA类型：质粒。质粒是小的环状双链DNA，它能通过超螺旋折叠来减小其体积，因此很容易通过细胞膜上的小孔。质粒含有几个值得提到的重要区域。商业化的质粒上有多个克隆位点，简称MCS。这个区域含有可被限制性内切酶识别并切割的特殊序列。当使用同样的限制性内切酶切割质粒和DNA片段后，研究人员就可将目的DNA片段插入到这个多克隆位点。
热激反应
开始热激转化前，要清洁工作台并保证所有的设备都已灭菌。要确定有足够的培养液和琼脂板，他们将为您所制备的感受态细胞提供营养。并且要确保高压灭菌所有的溶液。使用前，将液体培养基冷却到室温。并冷却琼脂培养基到50-55度，然后加入抗生素，倾倒制板。让平板在室温下得到凝固。
操作细菌时，应当时刻使用无菌技术来保证无菌。通常这要使用到本生喷灯来给器具或试剂灭菌，并用来形成一个空气对流 — 让空气中的污染物远离工作台。
热激转化反应开始前，预热培养液和含抗生素的LB琼脂板到37度，并且将水浴调至42度。接下来，在冰上融化感受态细胞。加1到5微升1个纳克每毫升预冷的质粒溶液到感受态细胞中，轻轻混匀。然后将细胞和质粒的混合物在冰上放置30分钟。到时间后，将感受态细胞和质粒的混合物置于42度水浴中热激30秒。将混合物从42度水浴中取出，立即置于冰上，并加入450微升培养液。然后置于37度摇床上，225rpm震荡培养1小时，使细胞得到恢复。
使用合适的无菌技术，吸取20到200微升菌液加到LB琼脂板上，用 细菌涂棒将其均匀涂在平板上。在37度温度下将LB平板倒置培养过夜，这样可以防止冷凝水污染细菌。
第二天，被质粒转化过的细菌将会形成菌落。接下来，计数菌落数目来计算转化效率，它是成功转化菌落的数目除以总DNA的量得到的数值。现在就可以挑取菌落做进一步的实验了。
制备感受态细胞
将要被制备成感受态的细菌被贮存在冻箱中。它们需先在冰上化冻，然后铺板到不含 抗生素的LB琼脂培养板上，37度过夜培养。
使用无菌操作，从琼脂板上挑取一个细菌菌落，接种到可多达500毫升的培养液中，37度摇床培养过夜。摇床可以防止细菌沉淀并可让培养液中的营养成分均匀分布。在细菌培养期间，可配制0.1摩尔浓度的氯化钙溶液，和含15%甘油的0.1摩尔浓度的氯化钙溶液，高压灭菌后放凉。
测定吸光值来判断细菌是否到达了生长的对数中期，这是它们容易摄入DNA的阶段。一旦细胞到达了这个阶段，将它们放置到冰上，并且接下来的整个过程中都要一直将它们保存在冰上。然后将细胞分装到两个大的离心管中，4度离心，弃掉上清，再重悬到冷却的100毫升0.1摩尔浓度的氯化钙溶液中，冰浴30分钟。重复该步骤至少一次。反复的离心和重悬会清洗细胞。
最后一次清洗之后，使用50毫升含15%甘油的0.1摩尔浓度氯化钙溶液重悬细菌。现在这些细菌就处于化学感受态了。分装这些感受态细胞，向每个离心管加入50微升，并贮存在－80度，直至用于热激转化。
应用和变化
细菌转化有多种应用和变化。除了热激法，另一种常用的转化技术是电穿孔法。正如其名字所指，该技术使用电流在细菌胞膜上造成微孔，这样DNA可以从中穿过。
为便于筛选转化细菌，质粒通常含有一个编码β-半乳糖甘酶的基因来帮助筛选。当LB平板上含有这种酶的底物时，转化了含插入片段质粒的细菌会产生白色菌落，而那些没有被转化的则会产生蓝色菌落。这种方法叫做蓝白斑筛选。
大多数转化实验的目的是通过快速分裂的细菌获得大量携带目的基因的质粒。所以在细菌转化之后，下一步是在含有抗生素的液体培养基中大量培养该种细菌，然后进行质粒纯化，也就是从细菌中纯化质粒。有许多商品化的试剂盒可用于纯化质粒。
某些时候，转化的目的是通过细菌制备大量由质粒编码的蛋白质。这里您看到细菌细胞被匀桨和裂解，然后通过亲和纯化技术来分离目的蛋白。纯化得到大量蛋白质后，可以对蛋白质进行结晶，并进行结构分析。
结语
您刚观看的是JoVE对热激转化的介绍。本短片中，我们回顾了什么是热激转化，它是如何起作用，它的工作原理以及如何成功转化细菌。感谢您的观看。
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稀薄臂被打开后，为什么细胞内容物不出来，细胞外的超螺旋DNA可以进去？