Chapter 2. Molecular Genetics: Restriction Mapping of Plasmid DNA

Goals and Objectives

The purpose of this lab is to provide students the opportunity to learn some of the techniques that are central in the study of DNA. In lab you will be given two samples of DNA. One piece is from the lambda virus, DNA that is now completely known. The lambda is the standard to which you will compare your unknown. The unknown DNA is from a plasmid known as pMAP. To learn more about DNA, molecular biologists treat DNA with several restriction enzymes. As you know, the restriction enzymes will cut DNA only when there are particular base sequences. By cutting the DNA with several restriction enzymes, you will be able to determine the size of the DNA, the size of the DNA fragments and the arrangement of the cut sites.

Since the labs are limited to two hours, we will provide you with DNA that has already been treated with restriction enzymes. You will have five samples, and in lab you will run these samples using the electrophoresis units and determine the map of the DNA plasmid that these samples came from.

Procedure

You will begin class by loading the samples into the electrophoresis unit. The electrophoresis must run for at least 45 minutes. Once the samples are running, you can determine the bands of DNA by looking at a gel from one of the previous classes.

LOAD GEL

1. The gels will be stored in a container and will be in a buffer solution. Place the gel in the electrophoresis unit. Important: Place it such that the wells are nearest the black electrode. Handle the gel very gently, as it is very fragile.
2. Fill the electrophoresis box with TBE buffer (a blue solution) such that it just covers the entire surface of the gel.
3. You will use the micropipets to load the contents of each reaction tube into a separate well in the gel. Important: Use a clean micropipette for each solution.
4. Gently tap each container to get the DNA into the bottom of the container.
5. Draw the sample into the micropipette. It is best to squeeze it below the bulb.
6. Steady the pipette with two hands.
7. Dip the pipette tip through the surface of the buffer, position it over the well, and slowly expel the mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to punch the tip of the pipette through the bottom of the gel. It is a bit like “placing” the solution directly over the well and then gently expelling the contents. The DNA should be visible in the well.
8. Important: Carefully record what solutions are placed in each well. For this example we suggest the following order starting nearest the black electrode. Write the actual order on the diagram provided.
9. Close the top of the electrophoresis chamber and connect the electrodes to the power supply (black to black, red to red). Make sure both electrodes are plugged into the same channel (one on top of the other). Plug in the power supply and adjust to the following settings: set range on “low,” voltage to 155 volts.