Purpose
Continuing from last week, you now have isolated the plasmid DNA from the unknown bacterial strain. You know it's one of four plasmids your company uses; but need to know for sure what the plasmid identity is. In this exercise, you will make a restriction map of the unknown plasmid. You will run restriction endonuclease reactions on the DNA you isolated last week, analyze the results by running an agarose gel to separate the fragments, and by comparing your results to a reference DNA ladder, you will estimate the length of your digested DNA fragments. This will allow you to construct a map of the restriction cut sites. You will check your map against the four possible known plasmid maps to identify your unknown plasmid. Finally, you will complete the map by adding location numbers to the restriction sites based on the sizes of the DNA fragments from each cut.
Objectives
Upon successful completion of this lab, you will be able to:
- Conduct a restriction digestion reaction.
- Prepare agarose gels and load DNA samples.
- Analyze DNA fragments and determine the molecular weight of the fragments.
- Draw a partial restriction enzyme map.
Pre-lab Preparation
Read the assigned reading completely.
Outline the purpose, objectives, and procedures sections in your notebook.
- For the restriction digests:
- Calculate the volumes of reagents based on your DNA sample.
- List the possible single enzyme digests and double digest reactions (ten total)
Complete the pre-lab quizzes at least 24 hours before lab begins.