The MiniOne TM Reagent Kit: Electrophoresis 101

Transcription

1 The MiniOne TM Reagent Kit: Cat#: M3001

2 2 3 Reagent Kit Components Other Required Materials Laboratory Safety Experiment Procedures Teacher s Guide Student Worksheet Ordering Consumables Table of Contents Page Reagent Kit Components Ready-to-Load Samples MiniOne DNA Marker Unknown DNA 1 Unknown DNA 2 Dye 1 Dye 2 Dye 3 Supplies 10x 1% Agarose GreenGel in-a-cup Concentrated 20x Sodium Borate (20x SB) running buffer Microcentrifuge tubes Micropipette tips MiniOne TM Electrophoresis System Running tank Carriage Base Photo hood 42V Power Supply Conical flask MiniOne TM Casting System Casting stand Gel trays Gel combs Required Materials Other Materials Micropipette Microwave Oven Digital Camera or Cell Phone Camera Ruler Laboratory Safety 1. Exercise caution when heating and/or melting reagents. 2. Exercise caution when working with electrical equipment. 3. Gloves and eye protection should be used whenever possible, as a part of good laboratory practice. 4. Always wash hands thoroughly after handling biological materials or reagents.

3 4 Teacher s Guide Teacher s Guide 5 Teacher s Guide Note: This guide is provided to give sample answers to questions and to explain certain steps in the protocol. There may be other correct answers that are not included. Since there are variations in data, students should carefully analyze their data and justify their conclusions based on them. Experiment Objective To develop an understanding of electrophoresis principles. To analyze results and to calculate the sizes of unknown charged molecules from given information and experimental data. Background Looking at a sample of green dye, how can you know if it is really green? Could it be a mixture of blue and yellow dyes? Electrophoresis is a technique used in many areas of science to analyze and separate samples by applying a constant electric field. Biologists or forensic scientists may use this concept to separate mixtures of DNA or dyes into each component. The gel in gel electrophoresis can be made of agarose, which forms a matrix, through which sample particles attempt to move, as the electric field pulls them forward. Like an obstacle course, longer and bigger particles have a harder time moving, while shorter and smaller ones can race on through. Therefore, both particle size and their charge can affect the results of a gel electrophoresis experiment. Today, you are going to try this technique out to analyze various samples. You will be given a sample with a mixture of known DNA sizes and a sample of known dye sizes. Based on the migration results of the known samples, you can figure out the sizes of unknown mixtures by drawing a standard curve. Experiment Overview 1 Peel & microwave 30 sec 4 Load samples + 5 Run the gel 2 Cast the gel 3 Add running buffer & gel to buffer tank - 6 Record an image

4 6 Teacher s Guide Teacher s Guide 7 Note: This kit provides enough materials to run 10 Before the Lab experiments. The following suggested preparation steps assume that you are preparing to run all 10 experiments. Prepare Running Buffer 1. The kit provides 130 ml of concentrated 20x Sodium Borate (20x SB) buffer. Dilute 1 volume of the concentrated 20x SB buffer with 9 volumes of DI water to get a 2x SB working concentration. Estimate that each experiment will need 125 ml of running buffer, plus extra in case of spills. Example calculation: (for 10 runs plus extra) Final volume: 1300 ml 20x SB needed: 1300 ml / 10 = 130 ml DI water needed: 1300 ml 130 ml = 1170 ml Note: 20x SB is equivalent to M boric acid with M sodium hydroxide 2. Following the calculation, dilute 130 ml of 20x SB with 1170 ml of DI water to get 130 ml of 2x SB for running buffer. 3. Optional: Aliquot 125 ml of diluted SB running buffer for each group of students before class starts, using the provided conical flasks. Prepare Sample Aliquots 1. The kit provides a total of 120 μl for each sample. 2. With the provided microcentrifuge tubes, aliquot 12 μl per sample for each group. There are a total of 3 DNA samples and 3 dye samples. Other Materials (Each group will need) 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 6 sample aliquots 125 ml of diluted SB running buffer, in the conical flask 1 micropipette & 6 pipette tips Part I: Electrophoresis Materials 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 6 samples Diluted SB running buffer 1 micropipette & 6 pipette tips Photo hood Well Sample Name Loading Volume 1 Dye 1 10 μl 2 Dye 2 10 μl 3 Dye 3 10 μl 4 MiniOne Marker 10 μl 5 Unknown DNA 1 10 μl 6 Unknown DNA 2 10 μl Procedure 1. Assemble MiniOne casting system. Place the casting stand on a level surface. Place gel trays in the two cavities of the casting stand and insert the comb into the slots at the top of the casting stand, such that the 6 well side is facing down. 2. Partially peel away the seal of a Agarose GreenGel in-a-cup and microwave for 30 secs. Slowly pour the hot gel solution into a gel tray. Check to see that there are no air bubbles in the gel solution. Let the agarose gel harden and turn opaque, approximately 30 mins. (DO NOT touch or move your gel until time is up.) Note: If melting multiple cups together, still microwave for 30 secs. Then check that each cup is completely melted. If not, add time in 5 sec increments until fully melted. Air bubbles in the gel solution, especially near the wells, can make it hard for DNA to migrate evenly (if there are bubbles, you can remove them with a pipette tip or a toothpick). If the gel is disturbed before it fully hardens, the gel can break or solidify unevenly, making it harder for DNA to migrate evenly. 3. Meanwhile, assemble the MiniOne electrophoresis system. Place the running tank into the black cavity of the carriage. With the included power supply, plug the carriage into the wall outlet. (DO NOT turn the power on yet.) 4. After the gel has hardened, carefully remove the comb. Then, remove the gel with the gel tray from the casting stand. Note: If you are having trouble removing the comb, pour some SB buffer on top of the gel and then completely pull the comb out. 5. Place the gel with the gel tray inside the running tank. Make sure that the wells are towards the negative end. (DO NOT turn the power on yet.)

5 8 Teacher s Guide Teacher s Guide 9 6. Measure out 125 ml of diluted SB running buffer and pour over your gel. There should be enough running buffer to fill both reservoirs of the running tank, plus a little over the top of the gel. Note: DNA is negatively charged, so to move the DNA into the gel with electricity, the DNA needs to be loaded on the negative side. It will then move towards the positive. What do you think the SB buffer is for? SB conducts electricity and completes the circuit so that DNA can be separated. 7. Turn the low intensity lights on by pressing the button on the carriage to help visualize the wells. Load your DNA samples into the wells, using 10 μl per well and changing to a new pipette tip each time. Remember to keep track of which samples you loaded in which wells. 8. Run that gel! While being careful not to bump your gel too much, make sure that the power cord is completely plugged in and then press the power button. The green LED next to the power button should turn on. Do you notice bubbles coming out from the electrodes? Note: How can you tell your gel is running? It bubbles at the electrodes. This is a redox reaction, forming H2 gas at the negative electrode and O2 gas at the positive electrode. 9. Observe the gel running for a few minutes. Then place the orange photo hood on top. The blue light allows you to see the DNA as it is running, and the orange photo hood helps filter excess light to improve contrast. 10. Allow the gel to run approximately mins or until DNA separation is sufficient. After your run is complete, turn off the power by pressing button. Use the photo hood and either your cell phone or camera to take a picture of the DNA. Then, remove the gel from the running tank and use a paper towel to gently dry the tray bottom. Place the gel tray on a piece of white paper and take another image to see the dyes. For both images, make sure to include a ruler so that you can measure migration distance. (Be sure to wipe off condensation from the inside of the hood, if any). Part II: Results What does your gel look like? Record images of the gel. Sample Results Part III: Analyze Your Data 1. According to your data, how many bands were resolved from each of your 6 samples? For the dye samples, note the color of each band you resolved. Dye 1: 1 band (orange), Dye 2: 2 bands (orange & light blue), Dye 3: 3 bands (orange, purple/dark blue & light blue), MiniOne marker: five bands, Unknown DNA 1: 1 band, Unknown DNA 2: 1 band 2. What electrical charge did your samples carry? How do you know? All the samples have components that are negatively charged. You know because they all move towards the positive end when the power is turned on. If there was anything positively charged then they would migrate towards the negative end and can run out the top of the gel. Part IV: Draw a Standard Curve Looking at your gel, Dye 1 should have 1 band, and Dye 2 should have 2 bands. The orange bands are from a dye called orange G, while the light blue band is from xylene cyanol. Dye 3 is an unknown mixture. The DNA marker contains a mixture of known DNA sizes. A table is given with the sizes of known dyes and DNA. Measure and record the migration distance of these known bands. With the distance and the known sizes, you can then construct a standard curve. Based on the standard curve, you can approximate the size of the unknowns from their migration distances. Knowns Dye/DNA Color Size (base pairs) Migration Distance Xylene Cyanol Light blue mm Orange G Orange 70 27mm MiniOne Marker Green Under blue light Unknowns mm mm mm mm mm Dye/DNA Color Size (base pairs) Migration Distance Dye 3 Light blue mm Dark blue mm Orange 70 27mm Unknown DNA mm Unknown DNA mm

6 10 Teacher s Guide Student Worksheet Using the images you took of the gel, measure the distance of the known bands relative to the positive edge of wells. Fill in the table. Note: Always measure the migration distance from the same point so that the distances are relative. By convention, measure the distance from the positive edge of the wells, since this is where dyes and DNA begin to enter the gel. Ex. measure from here to here 2. Use the filled in table to construct a standard curve on the semi log graph below. Distances are plotted linearly on the x-axis and base pairs are plotted on the log scale on the y-axis. Plot the graph for the knowns. Then draw a best-fit line based on your data. Sample Graph: y 3. Measure the migration distances for the unknown bands. Use the standard curve to extrapolate the sizes of the unknowns. Record the results in the table above. x Experiment Objective Background Looking at a sample of green dye, how can you know if it is really green? Could it be a mixture of blue and yellow dyes? Electrophoresis is a technique used in many areas of science to analyze and separate samples by applying a constant electric field. Biologists or forensic scientists may use this concept to separate mixtures of DNA or dyes into each component. The gel in gel electrophoresis can be made of agarose, which forms a matrix, through which sample particles attempt to move, as the electric field pulls them forward. Like an obstacle course, longer and bigger particles have a harder time moving, while shorter and smaller ones can race on through. Therefore, both particle size and their charge can affect the results of a gel electrophoresis experiment. Today, you are going to try this technique out to analyze various samples. You will be given a sample with a mixture of known DNA sizes and a sample of known dye sizes. Based on the migration results of the known samples, you can figure out the sizes of unknown mixtures by drawing a standard curve. Part I: Electrophoresis Materials 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 6 samples Diluted SB running buffer 1 micropipette & 6 pipette tips Photo hood Student Worksheet To develop an understanding of electrophoresis principles. To analyze results and to calculate the sizes of unknown charged molecules from given information and experimental data. Well Sample Name Loading Volume 1 Dye 1 10 μl 2 Dye 2 10 μl 3 Dye 3 10 μl 4 MiniOne Marker 10 μl 5 Unknown DNA 1 10 μl 6 Unknown DNA 2 10 μl Procedure 1. Assemble MiniOne casting system. Place the casting stand on a level surface. Place gel trays in the two cavities of the casting stand and insert the comb into the slots at the top of the casting stand, such that the 6 well side is facing down. 2. Partially peel away the seal of a 1% agarose gel cup and microwave for 30 secs. Slowly pour the hot gel solution into a gel tray. Check to see that there are no air bubbles in the gel solution. Let the agarose gel harden and turn opaque, approximately 30 mins. (DO NOT touch or move your gel until time is up.)

7 12 Student Worksheet Student Worksheet Meanwhile, assemble the MiniOne electrophoresis system. Place the running tank into the black cavity of the carriage. With the included power supply, plug the base into the wall outlet. (DO NOT turn the power on yet.) Part II: Results What does your gel look like? Record images of the gel. 4. After the gel has hardened, carefully remove the comb. Then, remove the gel with the gel tray from the casting stand. 5. Place the gel with the gel tray inside the running tank. Make sure that the wells are towards the negative end. (DO NOT turn the power on yet.) 6. Measure out 125 ml of diluted SB running buffer and pour over your gel. There should be enough running buffer to fill both reservoirs of the running tank, plus a little over the top of the gel. 7. Turn the low intensity lights on by pressing the button on the carriage to help visualize the wells. Load your DNA samples into the wells, using 10 μl per well and changing to a new pipette tip each time. Remember to keep track of which samples you loaded in which wells. 8. Run that gel! While being careful not to bump your gel too much, make sure that the power cord is completely plugged in and then press the power button. The green LED next to the power button should turn on. Do you notice bubbles coming out from the electrodes? 9. Observe the gel running for a few minutes. Then place the orange photo hood on top. The blue light allows you to see the DNA as it is running, and the orange photo hood helps filter excess light to improve contrast. 10. Allow the gel to run approximately mins or until DNA separation is sufficient. After your run is complete, turn off the power by pressing button. Use the photo hood and either your cell phone or camera to take a picture of the DNA. Then, remove the gel from the running tank and use a paper towel to gently dry the tray bottom. Place the gel tray on a piece of white paper and take another image to see the dyes. For both images, make sure to include a ruler so that you can measure migration distance. (Be sure to wipe off condensation from the inside of the hood, if any). Part III: Analyze Your Data 1. According to your data, how many bands were resolved from each of your 6 samples? For the dye samples, note the color of each band you resolved. 2. What electrical charge did your samples carry? How do you know? Part IV: Draw a Standard Curve Looking at your gel, Dye 1 should have 1 band, and Dye 2 should have 2 bands. The orange bands are from a dye called orange G, while the light blue band is from xylene cyanol. Dye 3 is an unknown mixture. The DNA marker contains a mixture of known DNA sizes. A table is given with the sizes of known dyes and DNA. Measure and record the migration distance of these known bands. With the distance and the known sizes, you can then construct a standard curve. Based on the standard curve, you can approximate the size of the unknowns from their migration distances.

8 14 Student Worksheet Using the images you took of the gel, measure the distance of the known bands relative to the positive edge of wells. Fill in the table. Knowns Dye/DNA Color Size (base pairs) Migration Distance Xylene Cyanol Light blue 2800 Orange G Orange 70 MiniOne Marker Green Under blue light Unknowns Dye/DNA Color Size (base pairs) Migration Distance Dye 3 Unknown DNA 1 Unknown DNA 2 2. Use the filled in table to construct a standard curve on the semi log graph below. Distances are plotted linearly on the x-axis and base pairs are plotted on the log scale on the y-axis. Plot the graph for the knowns. Then draw a best-fit line based on your data. y Ordering Consumables Call (858) or (800) Cat # Item Name Description M3001 Kit Basics of electrophoresis, for 10 groups. M3002 M3003 How to Pipette & Practice Gel Loading Kit PTC- To Taste or Not To Taste Kit Learn to pipette and practice fine motor control, for 20 groups. Relate genetics and electrophoresis as a tool, for 10 groups. M3004 DNA Fingerprinting Kit Examine genetic variations, for 10 groups. M3102 M3101 1% Agarose GreenGels in-a-cup Concentrated 20x Sodium Borate Buffer Easily cast gels to adapt your experiments, 10 gels/pk. Just dilute and it is ready to use. M3104 MiniOne Marker Load next to samples for easy comparison 5 bands, 500 μl for 50 loads M3107 M3108 M3109 M3110 M3111 M ml Microcentrifuge Tubes, Clear 0.6 ml Microcentrifuge Tubes, Rainbow 1.7 ml Microcentrifuge Tubes, Clear 1.7 ml Microcentrifuge Tubes, Rainbow μl, Micropipette Tips, Clear 1-10 μl, Micropipette Tips, Clear For sample storage, 200/pk For sample storage, 200/pk For sample storage, 200/pk For sample storage, 200/pk For use with micropipette, non-sterile, 250/pk For use with micropipette, non-sterile, 250/pk x 3. Measure the migration distances for the unknown bands. Use the standard curve to extrapolate the sizes of the unknowns. Record the results in the table above.

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