NAME: ______
PREPARING DNA SAMPLES AND RUNNING A GEL
PURPOSE
To learn the gel electrophoresis technique.
INTRODUCTION
DNA can be analyzed using the gel electrophoresis technique. When an electric field is applied across a gel, negatively charged DNA molecules will migrate through the gel matrix toward the positively charged cathode at a rate relative to size. An analyst determines the size of DNA fragments by the migration distance; smaller fragments will move farther through the gel than larger fragments. Before the DNA can be loaded onto the gel it first needs to be mixed with a loading dye. When current is applied, not only does the DNA move but so does the loading dye. Within five to ten minutes two or more distinct bands appear. These are markers that show the progress of the DNA down the gel. After the gel has been allowed to run it is then stained with a dye that adheres to the DNA due to its negative charge. Next, the DNA is ready to be put in a de-staining solution to remove excess dye. When the de-staining process is complete it is now time to view the gel. Distinct bands should appear when the gel is contrasted against a light source usually a gel lamp. At this point the DNA bands are ready to be measured and analyzed.
Now let’s try to run a gel!
MATERIALS
Latex Gloves 1 Microtube Crime Scene DNA
Safety Glasses 4 Microtubes of Suspect DNA
Loading Dye 0.5-10 uL Micropipetter
Gel Box 0.5-10 μL Micropipetter Tips
Microtube Rack Container for Tip Disposal
Microtubes λ DNA Ladder
Nucleostain 1 X TAE Buffer
Gel Lamp
PROCEDURE
1. Before beginning, be sure to glove your hands and don safety glasses.
2. Have a properly prepared gel box as described in earlier protocols.
3. In a microtube rack place:
Sample DNA
Microtubes containing suspect DNA
Microtubes containing crime scene DNA
Microtubes containing DNA ladder
Microtubes containing Loading Dye
6 empty microtubes
4. Using a permanent marker label 5 microtubes as follows: suspect A, suspect B, suspect C, suspect D, and crime scene. Label the microtube containing the DNA ladder as well.
5. Using a 0.5-10 uL micropipetter with a 0.5-10 μL micropipetter tip place 5 uL of loading dye in each of the five tubes marked for DNA. In the microtube containing the ladder add only 3 uL of loading dye.
6. Using a fresh tip, transfer 10 uL of suspect A DNA into the corresponding microtube containing the loading dye. Repeat this step with the remaining 4 suspect DNA samples.
Be sure to change tips between each suspect sample. We don’t want to mix DNA!
Helpful Hint: When adding the DNA to the loading dye it is important to mix the two solutions together. This can be achieved by pipetting the combined solution up and down 3 times.
7. Using a fresh tip load 3 uL of λ DNA ladder into the tube marked for the ladder. Be sure to mix the DNA with the loading dye by using the above technique.
Stop and read step 8 carefully!
8. Loading a well is a very delicate process. Follow the steps described below to ensure the wells are not damage during loading.
· Pull up the solution you wish to load and begin transferring into the well by slowly and carefully placing the tip inside the well.
· Feel for the edges of the gel gently by moving the tip back and forth.
· Do not poke the pipette tip into the bottom or the sides of the gel.
· Slowly expel the DNA/loading dye solution into the well. Press the pipette button only to the first stop. There will be solution remaining in the tip.
The second stop on a micropipetter expels a burst of air which serves to push the last drop of liquid out of a tip. This burst of air is strong enough to break a well. For a successful gel it is not important to expel this last drop of solution.
· Slowly pull the pipette tip out of the well but do not release the plunger. This can cause the sample to be sucked back into the pipette tip.
Be sure to change tips between each suspect sample. We don’t want to mix DNA!
9. Using a 0.5-10 uL micropipetter with a 0.5-10 μL micropipetter tip, load 6 uL of the ladder-dye mix into the first well.
Remember to change tips between each sample.
10. Load 10 μL of the DNA samples in the next 5 wells.
Remember to change tips between each sample.
11. Place the top of the gel box onto the gel tray. Make sure that negative (black) leads connect with negative and positive (red) leads connect with positive.
12. Plug the box into a power supply by matching the negative plug with the negative input slot and the positive plug with the positive input slot.
13. Set the Volts at 75 V, set the Amps to 185 A and set the time for 90 minutes. Make sure that the K button (second button down) on the box is reading for Volts and not Amps. After checking the settings again, press the run button.
Helpful Hint: if everything is working correctly, little bubbles from the electrodes should appear in the well.
14. After a few minutes check to see if the DNA sample is running from Negative (black electrode) to Positive (red electrode). Distinct blue and purple bands should appear within 5 to 10 minutes. This is the loading dye and can be used as a marker to follow the progression of the DNA.
15. When the gel is finished running, turn the power supply off and remove the lid from the gel box.
16. Gently take the gel out of the box and place it in a plastic container. Add Nucleostain until the gel is completely submerged. Place a lid over the container and let the gel soak overnight.
17. Once the gel has been allowed to stain overnight put on gloves and remove the gel from the staining container. Place the gel in an empty container and add 1 X TAE buffer until the gel is submerged.
Helpful Hint: Nucleostain may be poured back into its original bottle and reused.
18. Remove the gel from the plastic container and place it on the gel lamp. Now the gel may be analyzed.
19. The distance that each DNA fragment moves is
used to calculate the Rf factor, an abbreviation for “Relative Mobility of the Fragment”. The Rf value can then be used to calculate the number of base pairs in the fragment.
Calculate the Rf factor for each fragment using the equation:
Rf = distance in mm that the DNA fragment migrated
distance in mm from the well to the dye
Graph the class’s data for analysis. Include appropriate labels.
QUESTIONS AND CONCLUSIONS
1. What special property of DNA allows for gel electrophoresis and staining adhesion?
2. Describe two precautions you take when preparing and running a gel?
3. Why is a ladder loaded onto a gel?
4. After examining your graph, what conclusions can you draw regarding the size of the class’s DNA fragments? What can be concluded regarding the restriction enzymes used and the cutting process?
5. In addition to DNA analysis, name at least two other applications in which gel electrophoresis could prove helpful.