如何根据230nm289nm260nm的吸光度判断已提取纯化的DNA的浓度和纯度

2025-03-16 07:09:30
推荐回答(1个)
回答1:

紫外吸收检测DNA浓度与纯度2007年11月13日 星期二 11:40目的: 了解紫外吸收检测DNA浓度与纯度的原理,掌握测定方法。原理: 核酸的最大吸收波长为260nm,蛋白质为280nm,在波长260nm时,1OD值相当于双链DNA浓度为50μg/ml,单链寡核苷酸的含量为30μg/ml,可以据此来计算核酸样品的浓度,还可通过测定在260nm和280nm的OD值的比值(OD260/OD280),估计核酸的纯度。纯净DNA的比值为1.8, RNA为2.0。若比值高于1.8说明DNA样品中的RNA尚未除尽,若样品中含有酚和蛋白质将导致比值降低。270nm存在高吸收表明有酚的干扰。 紫外分光光度法只能用于测定浓度大于0.25μg/ml的核酸溶液,对浓度更小的样品,可采用荧光分光光度法。试剂与仪器: 提取的质粒DNA,紫外分光光度仪。操作步骤:1) 分光光度计先用水在260nm和280nm两个波长下校零。2) 取质粒DNA样品2μl,用水稀释100倍,转入分光光度计的石英比色杯中。3) 在260nm和280nm分别读出样品光密度值。如果样品浓度单位为μg/μl,则样品DNA浓度为OD 值的10倍,即如OD260=0.1,则样品浓度即为1μg/μl。4) 若OD260/OD280大于1.8,说明仍有RNA,可以考虑用RNA酶处理样品,若小于1.8,说明样品中含有蛋白质或酚,应再用酚/氯仿抽提,以乙醇沉淀纯化DNA。

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