Purpose
This experiment is the first of two experiments introducing essential DNA methods used by biochemists and molecular biologists. In the first week, you will isolate and measure the concentration of a small circular DNA plasmid from the bacterium Escherichia coli (E. coli). In the second week, you will characterize the plasmid by constructing a map of restriction enzyme sites. To isolate DNA from any organism, you must disrupt the cell wall or membrane, dissociate any bound proteins, and separate the DNA from other soluble compounds by taking advantage of the negatively charged nature of DNA. The isolated DNA can then be characterized and quantified by ultraviolet spectroscopy using the known extinction coefficient. Finally, we will check the quality of the purification by agarose gel electrophoresis visualized with ethidium bromide stain, a compound that specifically binds to nucleobases.
Premise
Imagine you work in a biochemistry lab. As is the common practice, your lab preserves important plasmids by transforming them into an E. coli stain and storing them at -80 °C. Anytime a researcher needs some plasmid DNA for cloning, they simply grow the bacteria containing the plasmid in a few milliliters of media and purify the plasmid. You go to the freezer to find a plasmid called pAB124, which you need for an experiment, but you cannot find it.
The pen labels have rubbed-off on several tubes in your freezer (a common problem with frozen samples). You grab a tube that you are pretty sure is the right one. But how do you know for sure? Over the next two labs you will solve this problem.
This week, you will plasmid DNA from a bacterial culture. Next week, you will digest it with restriction enzymes to build a plasmid map. You will check this map against the maps of known possible plasmids to identify the one in your tube. At the conclusion, you will complete a worksheet that summarizes the results and conclusions you will want to report to your supervisor.
Objectives
After this week's lab, you will be able to:
- Perform alkaline lysis plasmid DNA purification on bacterial cultures
- Quantify DNA via UV-Spectrometry and identify impurities
- Run agarose gel electrophoresis on purified DNA samples
- Understand the mechanism and inherent risks of ethidium bromide staining
Pre-lab Preparation
Read this lesson and the assigned reading completely.
Pass the prelab quiz with at least 70% by midnight before your lab.
Outline the objectives and procedures sections in your notebook. There is no post-lab assignment this week.