Molecular Rainbow Dye Electrophoresis Student Materials

Transcription

1 Dye Electrophoresis Student Materials Introduction. 2 Pre-Lab Questions 4 Lab Protocol. 5 Data Collection Worksheet 6 Post-Lab Questions and Analysis.. 7 Last updated: 10/23/2017

2 Introduction Take just a few seconds and answer this question for yourself: Biology is the study of living things, but how are living things studied? You are going to venture into the field of molecular biology by exploring one tool that allows the study of biology at the molecular level. Molecules like DNA, RNA and proteins are not living, but understanding these molecules informs us about living things. How can you study molecules of DNA, RNA or protein when they are too small to be seen, even with a microscope? A good first step may be to determine the size of a molecule. A common way to measure molecular size is by gel electrophoresis, which is a big name for a simple concept. Here is an analogy to explain gel electrophoresis. Imagine a very dense forest of trees where each tree is only one foot away from every other tree. You and a mouse must each run through the forest from point A to point B. You and the mouse begin at the same time. Which will reach the other side first, you or the mouse? Write your ideas here: The gel in gel electrophoresis is like the forest except instead of trees, it is a mesh formed of polymers. It is a lot like JELL-O, but for electrophoresis, the gels are made of agarose. Agarose is a carbohydrate, purified from seaweed, whereas JELL-O is mostly gelatin made from animal products like collagen. Once solidified, the gel is submerged in a tank filled with an electrophoresis buffer (a salt solution such as trisacetate EDTA) that controls the ph and conducts electricity. The molecules to be studied are suspended in wells in the gel, and an electric field is applied (see Figure 1). Figure 1. Illustration of gel electrophoresis equipment. Image Credit: In what direction will molecules with a negative surface charge move? In what direction will molecules with a positive surface charge move? 2

3 Molecules with a positive surface charge will move through the gel towards the cathode, the negatively charged electrode. Molecules with a negative surface charge will move through the gel towards the anode, the positively charged electrode. If the relative charges of the molecules are the same, then the molecules will move through the gel at different rates based on their size. The smaller fragments or molecules will move faster (see Figure 2). Figure 2. Dyes separated by gel electrophoresis. Dyes migrate away from the wells based on their overall charge and size. - electrode wells + electrode Image Credit: In this activity, you will use agarose gel electrophoresis and dye standards to determine what dye(s) are found in two mystery samples. 3

4 Pre-Lab Questions Directions: After reading through the introduction and protocol for the Molecular Rainbow lab, answer the questions below. 1. How does agarose gel electrophoresis work to separate molecules from one another? What is the force that causes the molecules to move? 2. What is the role of the electrophoresis buffer in this experiment? 3. You observe the following while performing gel electrophoresis on two molecules at the same time and under the same conditions. Although the molecules are the same size, you observe that molecule A barely moves from the well while molecule B moves 2 cm towards the positive electrode. Give at least one explanation as to why these two molecules of the same size behave differently in your experiment. Molecule A - electrode Molecule B + electrode wells 4. You observe the following while performing two different gel electrophoresis experiments of one molecule, molecule X. You run the two different gels for the same amount of time but you find that in one gel, molecule X moves 3 cm towards the positive electrode while in the other gel, molecule X moves 4 cm towards the positive electrode. Give at least two explanations as to why molecule X migrated a different distance in the same amount of time in the two experiments. 5. Explain why it is difficult to guess the mixture in unknown A or B by comparing the color of each to the known dyes. 4

5 Lab Protocol Materials: Check your workstations to make sure all supplies are present before beginning the lab. Student Workstation p20 micropipette and tips 1 microcentrifuge tube rack 5 standard dye samples 2 unknown dye samples 1 agarose gel (0.8%) with wells in the center 1 gel electrophoresis unit with power supply Procedure: Common Workstation 1X electrophoresis buffer 1. Prepare or source a 0.8% agarose gel and 1X electrophoresis buffer as instructed by your teacher. Check to make sure that the wells are in the center of the gel. 2. Assemble the gel box, and position it where it will run. 3. Using a p20 micropipette, load 10 µl of each of your samples into the gel wells in the following order. Tip: Be sure to use a fresh tip for each sample. Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 4. Place the cover on the electrophoresis box. Bromophenol Blue (BB) Methylene Blue (MB) Orange G (OG) Bromocresol Purple (BP) Allura Red AC (AR) empty Unknown A Unknown B 5. Connect the electrodes from the gel box to the power supply the red lead should be connected to the positive electrode and the black lead to the negative electrode. 6. Turn on the power supply, and set the voltage to ~125 volts. Check to confirm samples are moving away from the wells. Caution: Do not open the gel box lid while current is running through the gel box. 7. Run the gels at constant voltage for five minutes, and note what you see in the lanes for unknown A and B on the Data Collection Worksheet. 8. Continue to run your gel until the fastest moving dye is about two-thirds of the distance from the wells to the end of the gel about 15 minutes. Turn off the power supply, disconnect the electrodes, and carefully remove the gel from the box. 5

6 Data Collection Worksheet 1. After you ran the gel for five minutes, what did you see in the lanes for unknown A and B? Write your observations below. 2. After you have removed your gel from the electrophoresis chamber, use a straight edge to draw your results on the gel template below as accurately as possible. Label the positive and negative poles. If possible, take a photograph of your gel and attach it to this sheet. Sample: BB MB OG BP AR A B wells 3. Compare the bands seen in the lanes for Unknown A and Unknown B with the bands seen in lanes 1-5. Can you determine the mixture of dyes in each unknown? Unknown A = Unknown B = 6

7 Post-Lab Questions and Analysis Directions: After completing the Molecular Rainbow lab, answer the questions below. 1. You probably could not determine the mixtures in unknown A and B at five minutes, but you likely could determine the mixtures in unknown A and B at the end of your experiment. Explain why running the gel longer gave you more information. 2. You have a tube of purple dye that is either Purple X or Purple Y, but you do not know which. Assume you have all of the materials for the lab you just completed including stock solution of the following dyes. The size and charge of the dyes you used today are given in the shaded area of the table below. The size and charge of Purple X and Purple Y are given in the unshaded area of the table below. Biological dye ~ Formula Weight Charge Color in TAE Orange G Blue Methylene Blue Yellow Bromophenol Blue Blue Bromocresol Purple Blue Allura Red AC Red Purple X Purple Purple Y Purple Design a gel electrophoresis experiment that will allow you to determine if the unknown purple dye is Purple X or Purple Y. Remember that you have only one or the other, not both. Label the wells of the gel below with the samples you plan to run in each well. Try to use the fewest possible lanes to identify the unknown purple dye. Sketch what you think you would see on your gel after the electrophoresis experiment is complete. Describe the data you would obtain from the gel that would allow you to identify the unknown purple dye. 7

8 - electrode + electrode 8