RE Digestion of pGLO

BACKGROUND

As we have seen, agarose gel electrophoresis is a simple technique that allows researchers to separate DNA fragments based on their size. In this experiment, we will see how this technique, in combination with the use of restriction enzymes, can be used to determine important information about a DNA molecule. More specifically, we will digest the plasmid DNA you isolated previously with three different restriction enzymes. The resulting fragments then will be separated by gel electrophoresis. By analyzing the results, we will be able to determine the size of the plasmid and the relative position of the restriction sites within this plasmid.


GOALSA

Digest pGLO plasmid DNA with a series of restriction enzymes
Analyze the resulting DNA fragments by gel electrophoresis

MATERIALS

pGLO plasmid DNA
Pst I restriction enzyme
Hind III restriction enzyme
Eco RI restriction enzyme
2x RE buffer
100 bp DNA ladder (Bio-Rad)
5x loading dye

PROTOCOL


1. In separate microfuge tubes, set up five of the following six restriction enzyme digests:

2. Incubate in the 37 degree water bath for 1 hour.
3. After this incubation, add 2.5ul of 5x dye to each digestion.
4. Load the samples on a 1% agarose gel (in 0.5x TBE, with ethidium bromide). Load 5 ul of the 100bp ladder in each of 2 wells.
5. Run the gel at ~80 volts for 1 - 1.5 hours.

Questions:
What are the sizes of the fragments that you observe?
What is the size of the plasmid?
What are the relative locations of the Pst I, Hind III, and Eco RI restriction sites?
What additional experiment(s) could you do to address the previous question?