Gel Electrophoresis is a simple yet powerful technique that is essential to any biotechnology laboratory. Scientists use Gel Electrophoresis to separate out macromolecules such as DNA, RNA, and proteins based on size and charge. Clinical chemists use Gel Electrophoresis to focus on proteins, and molecular biologists and biotechnologists are more interested in DNA and RNA. This protocol focuses on Gel Electrophoresis with DNA.
DNA has a negative charge, so if it passes through an electric current, it’ll be driven towards a positive charge and away from a negative charge. Scientists take advantage of that in Gel Electrophoresis by suspending DNA into a gel and running a current through the gel apparatus, forcing the DNA to move. The DNA passes through the agarose gel matrix, and the longer and more complex the DNA, the slower it takes to make it through. Using this technique, biologists can determine the size and base pair length of the DNA samples. The DNA can also be purified for other labs, such as PCR, DNA sequencing, or Southern Blotting.
This lab is good practice for math, micropipette, and solution-making skills, and can be split into two lab sessions if time doesn’t permit the entire experiment to be completed in one class session.
Materials:
· Agarose Powder
· DNA Ladder
· DNA Samples
· 50X Electrophoresis Buffer (TAE or TBE)
· DNA Gel Stain (SYBR Safe or other dyes)
· 6X Electrophoresis Loading Dye
Supplies:
· 1.5 mL Microcentrifuge Tubes
· 100 mL Graduated Cylinder
· 250 mL Erlenmeyer Flask
· 500 mL Beaker
· Horizontal Gel Electrophoresis Apparatus
o Gel Tray
o Casting Gates
o Gel Comb
o Gel Electrophoresis Chamber with Lid
· Gel Electrophoresis Power Supply
· Balance and weigh paper or weigh boats
· UV Light or UV Illuminator
Protocol:
1. Place the Gel Tray in the Gel Electrophoresis Chamber so that the notches on the sides of both line up. Slide the Casting Gates into the slots at opposite ends of the Gel Tray.
2. Place the Gel Comb into the slot of the Gel Tray closest to the black cathode. It should slide in the notches of the tray and chamber.
3. Prepare your Electrophoresis Buffer. This protocol needs 500 mL of TAE Buffer, so use a 100 mL graduated cylinder to measure out 10 mL of the 50X TAE stock solution and add it to the 500 mL beaker. Add 490 mL of distilled water. See the calculations section below for details.
4. Prepare 40 mL of 1.0% agarose in 1X TAE buffer. See the calculations section below for details.
a. Weigh out 0.40 g agarose and add it to your Erlenmeyer Flask.
b. Add 40 mL 1X TAE Buffer you made in step 3 and swirl to mix.
c. Your agarose will not dissolve at room temperature, so it needs to be heated. Microwave the agarose in 30 second increments, swirling the flask gently between each heating.
d. Continue to heat until all the agarose has been dissolved.
CAUTION:
· The Erlenmeyer Flask will be very hot! Use personal protective equipment such as goggles and thick gloves or oven mitts, as the agarose can superheat and boil.
· Overflowing agarose can cause burns.
· Wait until the agarose stops boiling before removing it from the microwave.
5. Wait for the agarose solution to cool to 55°C.
6. Add your DNA stain. We will be using 10,000X SYBR Safe DNA stain. Using a 10 μL micropipette, add 4 μL of the SYBR Safe to the Erlenmeyer Flask and swirl gently to mix. See the calculations section below for details.
7. Pour the agarose gel into the Gel Tray set up in step 1 until the agarose comes within 2-4 mm of the top of the teeth of the comb.
8. Let the gel cool and solidify. This will take around 20 minutes. It will change from transparent to opaque, and will jiggle like gelatin once set.
9. Remove the comb by pulling it gently straight up and out of the wells.
10. Remove the casting gates by pulling them straight up.
NOTE:
· If your lab period is short, you can stop here. Wrap the gel tray in a wet paper towel, seal in a plastic bag, and place into the fridge until the next lab period.
11. Add 1X TAE Buffer to the Gel Electrophoresis Chamber. Fill both reservoirs at each end of the chamber and cover the gel by about 2 mm. At this point, the Gel Electrophoresis Chamber will be very full, so be careful not to spill!
12. You need to dye your DNA samples so that you can see the DNA migrate across the gel.
a. In a clean 1.5 mL microcentrifuge tube, add 10 μL of your DNA sample.
b. Add 2 μL of the 6X loading dye to the same microcentrifuge tube. Pipette up and down gently to mix thoroughly.
13. Now it’s finally time to load your samples.
a. In the first well, add 5 to 10 μL of your DNA ladder.
b. In the next few wells, add up to 12 μL of your dyed DNA samples from step 12. The amount you add may vary, as sample size is based on the size of the comb you used in step 2.
14. Close the top of the Chamber with the Gel Electrophoresis Lid, making sure that the red terminal interacts with the red electrode and the black terminal interacts with the black electrode.
15. Connect the Electrophoresis Chamber to the power supply, connecting the wires to the color coded ports on the front of the power supply, red to red and black to black.
16. Using the power supply, set it for a 15 - 30 minute run at 100 volts, and press the run button. As soon as power is running to the Gel Electrophoresis Chamber, you should see a curtain of bubbles at the black electrode. You can also see a much thinner curtain of bubbles at the red electrode.
17. Keep an eye on your gel as it runs. You may need to end the run prematurely if it looks like the bands are getting close to the end of the gel. If they fall off the gel, they’re gone forever. You can check the gel under a UV light. If they haven’t separated well, put the gel back into the chamber and continue to run.
18. Once the gel has finished running, turn off the power supply, remove the electrodes from the power supply, and carefully remove the lid.
19. Remove the gel and Gel Tray from the Gel Electrophoresis Chamber. Don’t remove it from the Gel Tray. It will be extremely slippery due to the TAE Buffer, so be careful. If it breaks or tears, be patient with yourself – this can be a hard technique to nail, and you’re learning!
20. Slide your gel off of the Gel Tray and on a dry paper towel or large weigh boat. Congratulations! You just cast and ran an agarose gel!
21. To see the bands in sharp clarity, shine a UV light over the gel. You should see the bands of the ladder and the DNA samples once UV light hits the DNA.
Results:
Now that you have a gel in your hand, it’s time to figure out what those bands mean. In the first lane you added a 1 kb DNA ladder. Each band on the ladder is a length of DNA. As the gel runs, the different lengths run faster or slower depending on how large the DNA strands are. The larger the DNA, the slower they’ll run. Look at the manufacturer’s paperwork that you got when you ordered the DNA ladder – this paperwork will tell you the sizes of each band on the ladder. If you can’t find it or it got thrown away, it should be available on the manufacturer’s website – simply google the name of your DNA ladder.
Match up your DNA unknowns with a “rung” on the ladder, and that will tell you how large the DNA strand is. For example, if your DNA strand matches with the 200 base pair “rung” on the ladder, then your DNA strand is about 200 base pairs long. Using this technique, you can now estimate how long your DNA strands are and create labs based around this concept! (Many high school and college students love doing a CSI Murder Mystery lab.)
Troubleshooting:
· My agarose leaks when I try to pour it in the Gel Tray.
o Put the casting gates in the freezer and add them to the chamber just before you add the agarose solution.
o Press firmly down on the casting gates a little more.
o Switch out your casting gates for a different set.
o Use tape or Parafilm instead of casting gates. Don’t forget to remove it before you run or the tape will melt, ruining your results.
o Test where you’re getting leaks by adding tap water to the center of Chamber and seeing where the water is coming out.
· I can’t cover my gel with TAE Buffer.
o Your gel is too tall. You’ll have to dispose of the old gel and try again, using a little less agarose solution. You don’t need to use all of it. The TAE buffer won’t be ruined if you touch it (even if you aren’t wearing gloves), so you can reuse it as long as you haven’t turned on the power supply to the chamber.
· It’s difficult to get the samples in the wells.
o Have patience with yourself as you learn this technique. This part is can be difficult for experienced lab workers. Anxiety and inexperience will make shaky hands, so breathe and be understanding to yourself as you learn. It’ll get easier with time.
o Special long tips are available that may help you load the samples easier. Search for “Gel-Loading Micropipette Tips”.
o Hold your right wrist steady with your left as you pipet the samples in. (Or vice versa if you’re left handed.)
o Go extremely slowly. The DNA will sink as you add it to the gel, so you don’t need to enter the entire well. As long as the very tip of the tip is in the well, you can add the DNA sample.
o Practice, practice, practice!
· I turned on the power supply and set it to run but I don’t see any bubbles.
o It sounds like your power supply might be broken or damaged. Check with a colleague to see if they see any bubbles. Wait a few minutes to see if the DNA bands migrate. If they don’t, contact your manufacturer for more help.
· My power supply won’t turn on.
o Did you plug it in? It’s not battery powered and needs to be connected to an outlet to work.
· The chamber is getting hot once it starts to run.
o Some warmth is ok, but if it’s getting hot, you’re in trouble. End the run immediately, wait for the buffer to cool a little, and run again at a lower voltage. Stay below 200 volts, and decrease by 25 volts if it’s still getting hot.
· It’s taking too long to run the gel.
o Try decreasing the amount of agarose you add in step 4. Make a 0.8% or 0.6% gel instead of a 1.0% gel.
o Try increasing the voltage in step 15 up to 125 volts or 150 volts. Be very careful, as the increased voltage can cause your gel to melt and the chamber to become hot.
o Both of these tricks will affect your final result by creating a more blurry line on the gel.
· My results are too blurry.
o Try increasing the amount of agarose you add in step 4. Make a 1.2% or 1.4% gel instead of a 1.0% gel. This will mean the gel will take longer to run but will create a crisper line.
· My DNA ran backwards and off the gel.
o You mixed the electrodes up either at the lid or at the power supply. Remember, red always matches with red, and black always matches with black.
· My DNA ran forwards extremely quickly and off the gel.
o Did you add 1X TAE Buffer to the Chamber or distilled water? Adding distilled water will make the gel run very, very fast and smear any bands that remain.
· I stopped the run too early and the bands are too close together.
o Place the gel tray back in the Chamber and run it for another 15 minutes. Remember to keep an eye on your gel so that the bands don’t fall off the end!
· I stopped the run too late and the bands fell off the end of the gel.
o Restart the lab from step 1. Sometimes you can get a few bands that haven’t fallen off yet, but if all the bands are gone, you can’t get them back.
· I can’t see any bands on the gel.
o You forgot to add the DNA stain in step 6 or you didn’t wait for the agarose to cool long enough and the DNA stain was destroyed by the heat.
· I can see the DNA ladder but not my DNA samples.
o The DNA samples are too low to show on a gel. Try again with a higher DNA concentration or more DNA sample to see if it helps.
· I tried troubleshooting but I’m still having trouble.
o You can either contact your manufacturer and ask for help or go online and google your problem. ResearchGate is an online forum filled with professionals who are equipped to handle any questions you have or problems that can arise.
Videos:
Making an Agarose Gel - https://www.youtube.com/watch?v=wXiiTW3pflM
Running an Agarose Gel - https://www.youtube.com/watch?v=U2-5ukpKg_Q
Calculations:
Step 3 – Making 1X TAE Buffer with the Dilution Equation C1V1 = C2V2
We want our buffer to go from a large concentration to a 1X concentration. We’ll be using 50X stock, so change your variables if your stock concentration or final volume is different.
C1 = 50X C2 = 1X
V1 = ? mL V2 = 500 mL
1. 50X • x mL = 1X • 500 mL
2. (x mL = 500 X•mL)/50X
3. x mL = 10 mL 50X TAE
4. 500 mL – 10 mL 50X TAE = 490 mL distilled water
So the solution is 10 mL 50X TAE and 490 mL distilled water.
Step 4 – Making 40 mL of 1.0% agarose gel.
This gel is 1.0% weight per volume. Change your variables if your target concentration or final volume is different.
1. (1.0 g Agarose/ 100 mL solution) = (x g Agarose / 40mL solution)
2. Cross multiply to get:
(1.0 g x 40 mL)/ 100 mL = 0.40 g Agarose
So we’re weighing out 0.40 g of Agarose for our gel.
Step 6 – Diluting 10,000X SYBR Safe to 1X with Dilution Equation C1V1 = C2V2
In order for the SYBR Safe DNA stain to work properly, it needs to be diluted down to 1X. A different DNA stain might have a different starting concentration and if you’re not making 40 mL of agarose solution, these variables will be different.
C1 = 10,000X C2 = 1X
V1 = ? mL V2 = 40 mL
1. 10,000X • x mL = 1X • 40 mL
2. x mL = (40 X•mL/ 10,000 X)
3. x mL = 0.004 mL SYBR Safe
4. 0.004 mL • (1000 μL/ 1 mL) = 4 μL SYBR Safe
So we are adding 4 μL SYBR Safe to the 40 mL agarose solution