Confirmation of Restriction and Ligation: Using Agarose-Gel Electrophoresis
Lesson 3 of 9
Objective: Students will be able to use a special staining technique which enables them to visualize DNA fragments in an agarose gel medium.
All of the lab investigations in this unit are based on nothing less than authentic Nobel Prize–winning science. Kary Mullis received the Nobel Prize for his discovery of the Polymerase Chain Reaction or PCR. Werner Arbor, Daniel Nathans and Hamilton Smith received the Nobel Prize for their work with restriction enzymes. Stanley Cohen, Paul Berg and Herb Boyer received the highly esteemed prize for making the first recombinant DNA molecule. Actually, the recombinant DNA molecule that students create during this lab series extends beyond the scope of these honored Nobel Laureates original work as it incorporates a gene from a eukaryotic rather than prokaryotic organism into a plasmid. It would be impossible to explain the effect these advancements and breakthroughs have had on the development of biotechnology, medicine, forensic science, and research in general however perhaps by experiencing the same triumphs as these giants the significance will become apparent.
In this laboratory investigation, we confirm that BamHI and Hind III have digested the original pKAN-R and pARA plasmids and the restriction fragments have been ligated together by DNA ligase.
The purpose of this lab is to provide evidence that we have recombinant DNA molecules. The plasmid samples: digested, undigested and ligated, and use electrophoresis to visualize the DNA fragments.
I begin this lab with a skill-building technique pre-lab in order to expose students to the gel electrophoresis apparatus and to analyze some of the properties of physics and electricity that govern this piece of equipment. I then continue as I have done in the previous labs by setting the context of the work we will be doing and providing background information as illustrated on SLIDES 2 thru 6 of the AMGEN Recombinant DNA Lab Series PowerPoint Presentation.
At the end of this segment of our prelab discussion I check for understanding using a Pre Lab Quiz as well as visit the community lab area in our laboratory classroom in order to walk students through staging their workspaces or "bench" as shown in this photo. As we move about the community lab area and either stage or review staging that has already been completed, I explain the significance of each piece of equipment and how it will be used in our work. Students record notes from our discussion in their laboratory notebook as oftentimes the information provided appears on our Post Lab Quiz and will be covered in our Conclusion Questions.
Plasmid samples from previous lab, pARA-R Restriction Digest: An Introduction to Plasmids and RE's
Ligated plasmid (LIG tube)
5x loading dye
1x SB (or 0.5x TBE)
DNA size marker (25 ng/uL)
EQUIPMENT & SUPPLIES
P-20 micropipette and tips
1.5 mL microfuge tubes
Plastic microfuge tube rack
Microcentrifuge or Table Top Centrifuge
1. Collect the five plasmid samples and the DNA marker from your teacher and place them in your plastic tube rack. You should have six tubes.
2. Obtain five clean 1.5 mL microfuge tubes and label them as follows: A-, A+, K-, K+, and L. The microcentrifuge tube with the marker should already be labeled.
3. The following reaction matrix summarizes plasmid sample preparation for gel electrophoresis.
4. Prepare the gel and electrophoresis box to receive these plasmid samples.
5. Take your plasmid samples and marker to the gel, along with your pipette and tips.
6. Unless your teacher has you load your samples in a particular sequence or pattern, be certain to record a drawing in your lab notebook which indicates the order your loaded your samples for future reference. See example here!
7. Using a clean tip, set your P-20 micropipettor to 10 μL. Aspirate 10 μL of your “DNA size marker” and slowly dispense it into the well.
8. Continue this procedure with the plasmid samples, following the order indicated on page 4.3. Change the tip for each sample. If you choose to load your samples in a different order, be certain to record the sample order in your notebook.
9. Close the gel box lid tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel (same side) with the negative (black) to negative (black) and positive (red) to positive (red).
10. On the power supply, set the voltage to 130-135v.
11. After two or three minutes, look at your gel and be certain that the purple dye (bromophenol blue) is moving toward to positive electrode. If it’s moving in the other direction—toward the negative (black) electrode—check the electrical leads to see whether they are plugged into the power supply correctly.
12. Be certain that you return your “LIG” tube to the front of the room. This tube should contain your recombinant plasmids and will be used for the next lab.
13. Your teacher will explain what to do with your gels, so listen carefully. If your lab time is short, you may not have sufficient time to complete the electrophoresis. The yellow dye will need to run just to the end of the gel, about 40–50 minutes.
The following photo illustrates the expected result for this laboratory investigation. I have annotated the result so that it can be used as both a training tool before the lab and perhaps as a control in which to compare the results received by instructors and their students.
To conclude this lab students individually complete the Lab 4 conclusion questions and prepare to share their insights during a whole group discussion. Since this lab includes the technique of gel electrophoresis which is a core practice in biotechnology, students complete an exit ticket in the form of a mock lab memo in which they have to explain to their pretend "employees" how to conduct a gel electrophoresis analysis! They must also explain to their staff how to interpret the Lab 4 results received and provide evidence for their interpretation!