MacGyvering Gel Electrophoresis: a Research-based Lab Lacey Howard Shearer Lab Research Group Sumrall High School Biology I Students Sorrell: 1st block 30, 3rd block 20 Hummell: 3rd block 21
5 E’s of the Lesson Plan Engage- ask students to discuss their knowledge of gel electrophoresis. Explore- have the students practice using pipettors with a scale and water. Elaborate- have the students perform their own gel electrophoresis experiment. Explain- discuss the principles of gel electrophoresis and how it is used in my research. Evaluate- have the students fill out lab worksheets with data results and summary questions.
State and National Subject Content Science as Inquiry Using the scientific method Science and Technology Distinguish and explain the applications of various tools and techniques used in DNA manipulation Steps in genetic engineering experiments Use of restriction enzymes Science in Personal and Social Perspectives Explain uses of technology in paternity testing and crime scene evaluations. Unifying Concepts and Processes
Materials Required Each gel electrophoresis kit needs: Five 9 V batteries connected in series Two alligator clips with one red wire and one black wire Small Tupperware with two holes on either end Two wires bent through holes in Tupperware and connected to alligator clips
Additional Materials 1X TAE buffer (Tris base, Acetic acid, EDTA) or 1x TBE buffer (Tris base, Borate, EDTA) Agarose Loading Dye Ethidium Bromide Safety glasses UV light Surgical gloves DNA Erlenmeyer flask Microwave
What is gel electrophoresis? Gel electrophoresis is a technique that separates pieces of DNA (or other biological molecules) by size so researchers can analyze them.
How does gel electrophoresis work? - Electrode Wells for DNA samples - Electrode + Electrode Side View + Electrode Top View
Agarose gel with Ethidium Bromide Staining DNA Ladder Larger DNA Fragments Smaller DNA Fragments
Molecular Weight Marker A mixture of different pieces of DNA of known size used to determine the size of fragments in the sample.
Staining DNA in the gel is invisible. To visualize DNA, gels are stained with ethidium bromide. A UV light is then used to visualize the EtBr. CAUTIONS: Ethidium Bromide is a carcinogen. wear gloves correct disposal UV light damages eyes wear goggles use photoimaging system
Ethidium bromide Ethidium bromide has a ring structure similar to the ring structure in DNA bases; therefore it intercolates between the bases. Shining UV light on ethidium bromide will cause it to fluoresce at visible wavelengths.
Loading Dye Purposes Components give color to sample give weight to sample so it will sink into well show progress of gel migration Components dyes (xylene cyanol, bromphenol blue, Orange G) weight (glycerol or ficoll)
Uses of DNA Gel Electrophoresis Is DNA present in sample? Is a PCR product present? Is the PCR product the correct size? Where in the DNA do specific enzymes cut? Also, a specific DNA fragment can be recovered from the gel.
Gel Electrophoresis Start to Finnish
Biology I Students Performing Electrophoresis
Changes for the Future This lab requires at least an hour of preparation time. To minimize the time students are waiting for the experiment to run without having work to do, give the lecture while the experiment is running. Explain more about PCR and restriction enzymes during the lecture. Engage students by talking about processes they see on crime scene shows like paternity testing and DNA evidence. Go over proper pipetting technique and aseptic technique before lab experiment.
References Desktop electrophoresis lab- moving molecules http://www.accessexcellence.org/AE/WWC.1993/moving.php DNA gel electrophoresis http://www.colorado.edu/Outreach/BSI/pdfs/Electrophoresis%20Notes.pdf
Q & A