概述
离心机是一种全球几乎所有研究实验室都在使用的仪器。离心是一个基本的实验室操作过程,它使用离心机来分离复杂混合物中的组分。在极高速度下离心实验样品,混合物中的组分会受到离心力的影响,使得较高密度的颗粒离开轴心方向移动,而密度小的颗粒则朝向轴心方向移动。这些颗粒会沉积在管底,形成所谓的沉淀物,这样分离得到的样品,或被称为上清的余液,都可用作进一步处理或者分析。
离心机的组成和离心的参数
离心机的主要元件是转头,它是高速离心时转动的部件。转头可以被固定在离心机上,也可以是一个离心机使用多个转头,将它们装在称为主轴的部件上。离心机转头通常还带有一个盖子,能紧紧拧上盖住,防止样品飞出来。许多离心机会有一个制冷单元,它可以保持内部温度在离心时得到控制。
离心机上还有旋钮或按钮用以输入每次离心所需要的参数:包括离心时间、温度、和离心快慢,它可以是以速度或以相对离心力为单位。离心速度用RPM来计量,即每分钟的旋转次数。RPM值有时有误导性,因为造成颗粒从混合物中分离出来的不是离心的速度,而是作用在这些颗粒上的作用力。这种作用力与离心机转头的半径相关,由于不同的离心机有不同的转头大小,这样在同一个RPM速度下,产生的作用力会有不同。
离心力也可以用RCF相对离心力来定量。RCF通常表现为地球重力加速度的倍数。RCF的计算公式是转头半径乘以角速度平方,然后除以地球的重力加速度。RCF通过下面的公式和RPM相关,其中r代表离心机的半径,以厘米为单位。对该公式的使用可能会影响到实验的成功或失败,但您不必在每个新的操作中都采用这种计算方法。离心机通常都附带了图表来帮助简单地换算RCF和RPM。用一个尺子连接离心机的半径和一个已知的RPM数值,就可以得到RCF数值。
离心操作
在开始离心您的样品之前,先要考虑温度。如果您在使用一个冷冻离心机,那么在开始前需要让仪器内部的温度达到您的理想值,或者用别的方法来给转头降温。
离心即将开始前,需确定您所有的管盖都盖紧不会松开了。在将离心管放入转头时,要保证每个样品和另一个相对位置的样品重量一致。如果您只有一个离心管,那就要找到另一个等重的离心管来保证平衡。如果您有三个同样重量的离心管,可以将它们摆成一个三角位置以保证相互平衡。重量平衡在离心中非常重要。离心机的转头在高速运动中有很高的动态能量。如果没有正确平衡好,整个离心单位会脱离它所在的位置,造成严重的损害。一旦您确定转头和盖子都固定好了,就可开始离心,在速度达到您设定的最高转速前不要离开。如果您发现问题,找一个有经验的实验人员来帮助您。
离心结束后,您应当会看见您的生物学样品位于离心管底部的沉淀中。它已经从称为上清的余液中分离出来了。可以通过倾倒,也就是直接倒出来去除上清,也可将上清吸出,就是用真空吸力将其吸出。纯化的样品可以被重新放回溶液中,这个过程称之为重悬。重复离心细胞,然后吸出上清留下细胞,再用缓冲液重悬细胞,这个过程称之为细胞洗涤。
现在您已经看到一些基本的离心技术,下面再来看一下一些离心机的类型以及您要进行的操作过程。
应用
固定角转头离心机可能是您在实验室里最常见到的。许多台式离心机就是这种类型。在这种离心机中,离心管呈固定的角度放置,它适用于差异离心方法。在这些方法中,可应用一系列不同速度的离心来纯化多种生物样品,例如动物细胞。通常这些方法会包括几个细胞洗涤步骤。
与固定角转头不同,甩平式转头有灵活的离心管支架,使得样品在旋转时向外甩起。这种转头对某些应用很有帮助,如密度梯度离心,可将生物样品在密度梯度介 质中分离到不同的密度层。这种离心有助于快速分离不同的细胞类型,或者分离某种单独的细胞器。
最后,超速离心机是实验室中所有离心机的老大哥。它的离心速度可以超过70,000RPM。这样它可以用来分离小颗粒,例如DNA或者病毒。因为这种离心机的高速度,要格外小心来保证它的样品承载得到了正确的平衡,并要确保转头和转头盖都是牢固的。
结语
您刚观看的是JoVE对离心机的介绍。本短片中,我们回顾了:什么是离心机以及它是怎样工作的;如何操作和使用离心机;一些安全措施和离心技术的不同应用。感谢您的观看,记得平衡您的离心管哦。
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