Lab 6: Molecular Biology

Other Links for this Lab Activity

Link to the AP Biology Transformation Lab Simulation

Transformation Lab Link Iowa State

Link to the AP Biology Gel Electrophoresis/Genetic Testing Lab Activity

University of Utah Genetics Science Learning Center
Electrophoresis Simulation

Gel Electrophoresis Simulation (Which Dog Did It?) Iowa State

Link to Lab Bench Lab Simulation (account and password required, then follow the links to lab bench)

Lab Six Review Essay

In the first part of the lab, you will use antibiotic-resistance plasmids to transform Escherichia coli. By introducing the gene for resistance to the antibiotic ampicillinfrom the plasmids into e-coli (e-coli is killed by ampicillin). If the e-coli incorporate the foreign DNA, they will become ampicillin resistant.

In the second part, you will use gel electrophoresis to separate fragments of DNA for further analysis. Then you will compare fragments of unknown size to fragments of a known size to calculate the unknown fragment sizes.

Genetic transformation occurs when a host organism takes in foreign DNA and expresses the foreign gene.

Bacteria have enzymes that cut, or "digest," the DNA of foreign organisms and thereby protect the cells from invaders such as viruses. These enzymes are known as restriction enzymes. Each is able to recognize and cut at a specific DNA sequence, known as a recognition sequence.

The discovery of restriction enzymes made genetic engineering possible because researchers could use them to cut DNA into fragments that could be analyzed and used in a variety of procedures.

Like all enzymes, restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences.

Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. The direction of movement is affected by the charge of the molecules, and the rate of movement is affected by their size and shape, the density of the gel, and the strength of the electrical field.

DNA is a negatively charged molecule, so it will move toward the positive pole of the gel when a current is applied. When DNA has been cut by restriction enzymes, the different-sized fragments will migrate at different rates. Because the smallest fragments move the most quickly, they will migrate the farthest during the time the current is on. Keep in mind that the length of each fragment is measured in number of DNA base pairs.

In this lab, you use plasmids that carry the amp^R gene to transform E. coli cells that lack this gene. Also prepare a second group of E. coli cells as a control to verify that E. coli will not grow on agar with ampicillin unless it is transformed.

The Process: (1) Transfer ampicillin sensitive E. Coli cells in their log phase of growth to cold CaCl₂solution. (2) Add amp^Rplasmids to the experimental cells only (not to control). (3) Heat-shock cells at 42 degrees C. (Some of the competent cells will take up the amp^Rplasmid and will be transformed). (4) Spread the treated cells on an agar plate containing ampicillin. (5) Incubate cells for 24 hours. (6) Experiment is finished. You should see that only colonies of E. coli that have been transformed by the amp^Rgene grows.

The procedure is the same for the control group except in step 2, where you add amp^R plasmids to the experimental cells but not to the control cells.

Three samples of DNA from a virus, the bacteriophage lambda. One sample will be uncut DNA, one will be incubated with the restriction enzyme HindIII, and one will be incubated with EcoRI. By separating the fragments of DNA by electrophoresis and staining the DNA for visualization you can determine the fragment sizes formed in the EcoRI digest.

Run the unknown DNA alongside DNA with known fragment sizes. The known DNA acts as a marker. In your laboratory, the DNA that has been cut with HindIII is the marker; you will use it to help you determine the fragment sizes in the EcoRI digest.

Making a standard curve for the known sample, DNA plus HindIII. Measure the distance each HindIII fragment migrated on the gel