做过分子实验的童鞋也许听说过甚至用过QIAGEN的产品,特别是那些用于DNA提取、提纯的试剂。QIAGEN是一家成立于德国的生物科技公司,主营分子生物实验试剂等等。 QIAGEN生产的DNA相关试剂有几十种之多,大致可以分为三大类: (1) DNA 提纯/清理试剂(DNA clean-up kit);(2)基因组DNA提取试剂(Genomic DNA kit);(3)质粒DNA提取试剂(Plasmid DNA kit)。小编在本文就简单介绍几个实验室常用的试剂。
DNA 提纯/清理试剂(DNA clean-up kit)
QIAquick PCR Purification Kit和QIAquick Gel Extraction Kit
显然,PCR purification kit是用来清理PCR反应液中可能剩下的蛋白质、阳离子和dNTPs,而Gel extraction kit则可以从琼脂胶中提纯DNA(电泳过后,提取指定条带)。两种试剂盒中提供的洗涤液和洗脱液基本相同,而且他们使用同一种硅胶滤柱(QIAquick column)。滤柱能够结合最多DNA的量是10 µg,最少使用30 µl 洗脱液,可提取的DNA片段的范围是100 bp-10 kb。
基因组DNA提取试剂(Genomic DNA kit)
QIAamp DNA Mini Kit
QIAamp 试剂盒可以用于提取人类组织细胞基因组DNA、线粒体DNA、细菌DNA、寄生虫DNA 和病毒DNA。不同于从PCR反应或者琼脂中提纯DNA,从活体中提取DNA要涉及到不同的细胞裂解方法,亦或是使用不同的缓冲液。详情还是请见试剂说明书。值得一提的是,DNA mini kit提供另一种硅胶滤柱(QIAamp column),基本反应原理和QIAquick 滤柱一样,DNA结合量在4-12 µg,但可以容下较大的基因组DNA。洗脱液使用量在50-200 µl。
质粒DNA提取试剂(Plasmid DNA kit)
QIAprep Spin Miniprep Kit
Spin Miniprep 试剂盒仅用来提取质粒DNA(High-copy plasmid, low-copy plasmid and cosmid DNA)。Miniprep 试剂盒所用的也是硅胶滤柱,名叫QIAprep column。滤柱最大DNA容量是20 µg,最少使用30µl 洗脱液。
上面所说的四种试剂盒,都是使用硅胶膜滤柱来吸附分离DNA的。基本原理如下图所示。在高盐环境中,盐阳离子打破硅胶负氧根和水之间的氢键,使得整体的硅胶携带正电,正好吸引携带负电的DNA分子。当DNA分子附着在硅胶上时,洗涤液可以将不能与硅胶结合的杂物分子洗掉。然后,使用低离子强度的缓冲液或者水,可以将DNA洗脱出去。加热过的洗脱液(<50°C)可以加速DNA从硅胶膜上脱离下来。不同的硅胶由于制作方法不同,会拥有不同的膜孔大小,所以可以拦截吸附不同长度的DNA分子。
上文所提到的四种试剂盒只是QIAGEN众多DNA处理试剂中最常见的几个,根据个人实验的需要,还有其他的试剂可以选择,比如Midiprep、Maxiprep可以一次性容下更多量的质粒DNA,加速反应流程。QIAGEN的DNA试剂基本没有操作难度,只要按照每种试剂的实验步骤一步一步来,还是很容易成功的。
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