使用溴化乙锭荧光法测定衣藻中的DNA和RNA。

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide.

作者信息

Valle O, Lien T, Knutsen G

出版信息

J Biochem Biophys Methods. 1981 Jun;4(5-6):271-7. doi: 10.1016/0165-022x(81)90067-1.

Abstract

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.

摘要

通过利用与核酸结合的溴化乙锭的荧光增强效应，设计出了一种用于快速、简便且灵敏地测定绿藻衣藻中DNA的方法。总荧光（DNA+RNA）通过在细胞裂解物中与溴化乙锭形成复合物来测定，细胞裂解物是通过将细胞样品与月桂酰肌氨酸钠、乙二胺四乙酸（EDTA）和氢氧化钠混合制成，然后在室温下孵育混合物5分钟，随后进行中和。为了测定DNA，将细胞样品在碱性裂解液中于60℃孵育45分钟，随后进行中和，再与溴化乙锭形成复合物，从而消化RNA。使用内部DNA标准校正由于细胞色素导致的荧光猝灭。