Question

You are working on a DNA sequencing experiment on bacteria, which requires you to extrac DNA from bacterial cells. Why does it make sense to include a step in the protocol that adds RNAase, an enzyme that degrades RNA, to your DNA extraction mixture?

Answer 1

The experiment is regarding DNA sequencing in bacteria. The primary step in the experiment is the extraction of DNA from bacterial cells. For this purpose there is a need of total cell DNA which is harvested in the suitable medium. Once we have the bacterial culture ready the next step is to centrifuge it which provides us with the bacterial pellet. The third step is cell extraction which is made possible by techniques of chemiccal lysis such as usage of lysozyme and EDTA. The end product of the cell extract is a mixture of nucleic acids(DNA, RNA) and proteins. Phenol chloroform method is a widely used method for extracting the nucleic acids from the mixture. In the method the cell extract is mixed up with phenol and chloroform in the ratio of 1:1. The mixture is then centrifuged. Post centrifugation nucleic acids(DNA,RNA) are found to be present in the upper aq medium and coagulated protein appeared in the interface. Carefully pipetting out the upper aq medium will provide us with only the RNA and DNA. But our aim is to extract DNA. Therefore, in order to get rid of RNA, RNase was added in the mixture which is a ribonuclease and rapidly degrades the RNA. The end product is thus DNA. This DNA can be purified further via ion exchange chromatography. The purified DNA is concentrated using ethanol precipitation. This is the whole protocol of DNA extraction.