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Nanodrop经验谈

发布: 2016-01-18 583阅读 112评论

Nanodrop是实验室中常见的分光光度计。与传统需要比色皿的分光光度计相比，Nanodrop所需样品的体积大大减少，只需要2 µL，同时也大大降低了实验准备以及清洗的时间。这些优势使得科研人员能够在一分钟内检测多个样品。当然如果你有更多的样品，还嫌效率不够高，你可以选择应用Nanodrop 8000系统，它能够同时检测8个样品。

1

Nanodrop能够在包括紫外及可见光区域在内的光谱范围内检测样品的吸光度信号。在实验开始前，需要在仪器的软件内设置检测样品的种类，包括蛋白质，DNA, RNA等。根据吸光度,能够计算样品的浓度。DNA及RNA 在260 nm处有最大吸收度，蛋白质在280 nm处有最大吸收峰值。浓度与吸光度的对应关系见下表。当然表中浓度与吸光度对应关系成立的前提是样品必须具有很高的纯度。样品纯度可以通过A260/280判定，其临界值可参考下表。 大家可能注意到，RNA的A260/280比值要比DNA的值高一些，这是因为碱基U的A260/280的比值要比碱基T的A260/280比值更高。

2

因此在利用Nanodrop检测核酸浓度的时候，除了A260/280外，A260/230也是检测样品纯度的一个指标，高纯度DNA和RNA的两个临界值同样是1.8以及2.0。A260/230在2.0-2.2之间是最为合理的数值。对于纯的核酸，A260/230的值通常大于A260/280的值。核酸提取过程中残留的有机物通常会对检测结果产生影响。如果检测结果显示230nm处具有很大的吸光值，样品很有可能被EDTA，碳水化合物或者硫氰酸胍等物质污染了。对Nanodrop检测结果产生直接影响的因素就是样品的纯度。A260/280是检测样品纯度的一个指标，但这个参数只衡量了蛋白质与核酸之间相互污染的情况。没有考虑DNA与RNA之间彼此污染以及其他物质污染的情况，比如苯酚在270 nm附近具有较大的吸光值。

小编想要说的最后一点是Nanodrop的检测范围。对于核酸来说，Nanodrop的检测浓度范围在2ng-15μg/μL之间。所以当样品的浓度很低时，比如从单个或少量细胞中提取的DNA或RNA，利用Nanodrop来测量浓度可能就不适合了。这时需要考虑其他的核酸定量方法比如荧光染料的方法。

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