Begin
Objective: Learn about the process of electrophoresis, walk through the steps of setting up a gel tank, casting an agarose gel, running a samples and DNA ladders.
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Welcome to Virtual Bioengineer:
Gel electrophoresis
© Amino Labs www.amino.bio
5. Analyzing Agarose Gell
What is a Electrophoresis?
What do we use it for?
4. Running Agarose Gell
3. Loading Gel with Samples
Electrophoresis is used to separate mixtures of DNA, RNA, or proteins according to their molecular size and charge.
The “agarose” in Agarose gel electrophoresis specifies the gel type used is agarose (as opposed to other types like Polyacrylamide).Agarose Gel electrophoresis is a tool/method for determining:DNA fragment sizeConcentration of DNAPurifying DNA
OK, Let’s Start
2. Preparing Electrophoresis Tank
1. Casting agarose gel
1. Casting agarose gell
A more in depth look at how electrophorsis works:
Because Nucleic acid polymers like deoxyribonucleic acid (DNA) have a negatively charged phosphate backbone, scientists realized they could cause these negatively charged molecules to move towards a positively charged electrode when a current was applied The negatively charged DNA backbone is pulled through the gel by electrostatic forces towards the cathodic electrode.As the nucleic acids are pulled toward the positively charged electrode, they bump into and get caught up by the agarose mesh. The larger the nucleic acid, the more it bumps into and gets hung up by the agarose molecules. This results in the large molecule moving more slowly toward the positively charged cathodeResults: Smaller DNA fragments move faster towards the positive charged cathode creating seperation of DNA depending on size!
Great!
Materials
Micropipette and Pipette Tips
Distilled Water
Agarose Powder
Agarose cast and combs
Start Making Agarose Gel
Microwave
Electrophorsis buffer
(TAE)
RedSafe (Florescent dye)
Nitrile Gloves
Distilled water is a type of purified water. Distilled water is water that has been boiled into vapor and condensed back into liquid in a separate container. Impurities in the original water that do not boil below or near the boiling point of water remain in the original container.
Approx 100 mL of water is needed to make an agarose gel.
Got it
Start Making LB Agar Plates
A micropipette is a common laboratory instrument used to measure small amounts of liquids with a volume range between 1 and 1000µl. A micropipette is also used to transfer a precise amount of fluid from one container to another.
For this experiment, you need a P10 micropipette and pipette tips.
You need a microwave to boil your water. Having boiled water will make sure your agarose powder will dissolve properly.
It is really important to make sure your water is fully boiling before you add your agarose!
TAE is a concentrated buffer solution used in gel electrophoresis tanks to create an optimal environment for the separation of DNA fragments. The solution contains Tris-acetate and EDTA, which help to maintain a stable pH and prevent degradation of the DNA during the electrophoresis process.
Agarose powder is a substance made from seaweed that is used in gel electrophoresis to separate DNA fragments. It forms a gel-like substance that allows the DNA fragments to be separated based on their size and charge.
A fluorescent dye that is commonly used as a safer and more sensitive alternative to traditional DNA stains, such as ethidium bromide, in gel electrophoresis. It binds specifically to DNA and emits bright red fluorescence under UV light, allowing for the visualization of DNA bands in the gel.
When doing science such as this you should wear gloves (latex, nitrile). You do this to protect you from your experiments and your experiments from you!
Gel casts are a container that you pour your molten agarose into so that it can cool down and solidify into the desired shape. Generally, the cast is made up of a plastic tray and 2 stoppers, often rubbers, to cap the ends of the tray so it can hold the molten liquid.
Click to Swirl for 5 seconds to mix!
Put on your purple Nitrile Gloves. You should always wear gloves to protect your experiments from you and you from your experiments
Get a clean, microwave-safe container like this bottle to hold 96 mL of distilled water.
Take your tube containing 4 mL of concentrated 25x TAE buffer. The 25x means that the concentration of the TAE is 25 times higher than you need for your experiment, so you dilute it in water to get 1x concentration, which is the concentration you want.
Add the 4 mL 25x TAE to the bottle.
Start by assembling the cast and comb that you will set your agarose gel in. Assemble the plastic tray and stoppers to form a rectangle that the molten agarose will be poured into.
To be able to add your DNA or samples in the gel, you will need to set some indents in it. These indents or holes are called wells. To make these wells, you will set a comb in the gel cast. The gel cast will typically have small slots on the sides to help guide you where to put the comb.
“Agarose is a refined sugar polymer that comes from agar! If you’ve made agar petri dishes before, you’ll be familiar with agar. Agarose is a purer from of agar, and you need agarose to make gels.
Take the tube of white powder that says agarose and pour it into your 1x TAE buffer in your mixing container.
Click to mix your agarose!
When making agarose, you’ll need to bring it to a boil. It’s important to loosen the lid on your water bottle before microwaving
Loosen the Lid
Time to Boil your Agarose
Your lid is loose so your bottle will not explode in the microwave!
Start your microwave with 1 minute on the timer. You will see boiling happen at around 45 seconds, but allow it to keep boiling until the minute is over. Compared to LB agar, agarose does not boil over very often.
Place your container in the microwave.
Click to mix your agarose!
Gently swirl the container for 5 seconds. Be careful, it will be hot!
To ensure your agarose is dissolved, microwave for another 15 seconds. If it looks cloudy, you can keep microwaving and swirling it.
Place your container in the microwave.
Great! Next
Your agarose is now boiling!
If you notice that some of the “bubbles”in your agarose do not act like normal bubbles in rising to the surface, that means they are not bubbles but are undisolved agarose. Be sure to keep microwaving and swirling the agarose until these disappear and your agarose is clear
Add the RedSafe to your agarose. RedSafe is a fluorescent dye that lets you see the DNA. You only require around 1 microlitre of dye so use a pipette!
** Do not forget this step or you will not see your DNA! **
Pour the gel
Great Job! You are now ready to poor your agarose into the cast.
Pour to here
Next Step
Pour
A standard gel requires 20 mL of molten agarose. You can measure 20-25 mL of your agarose and pour it into you cast or you can simply pour molten agarose into your cast until it is near the top (2-3 mm below the top).
Hold the pour button to fill the cast up to the marker
Retry
Thats too much agarose in the cast!
Time to Prepare the Tank
Allow your molten agarose to cool and solidify. The timing will depend on the temperature and humidity of your lab, but is typically around 15-30 minutes. You know your gel is set if it turns slightly white and is quite solid
you can leave the remainder in the container to microwave later on when you are ready to pour another gel.
First in a seperate container we’ll need to create 1x TAE buffer for the electrophorsis tan. This is different to the gel with cast - we won’t be adding any agarose
Pour 20 mL of concentrated 25x TAE into the large bottle with 480mls of distilled water to make a total of 500 mls.
2. Preparing Electrophoresis Tank
Remove the comb from your newly cast gel. Note that sometimes the agarose gel can stick and if you pull to fast the gel can rip. If this happens you need to cast a new one!
Carefully remove the comb from the gel.
Thats too much TAE buffer in the tank!
You want to barely submerge the gel in 1x TAE buffer. Once the gel is barely submerged in 1x TAE you should see the TAE enter into the wells.
Hold the button to fill the tank.
Fill Tank
Fill to here
Place your gel, still inside the casting tray, in the electrophoresis tank.
Most modern electrophoresis systems only allow you to place the gel in one orientation. However, if you can put it in two directions you want the wells closest to the (-) electrode with black wires and furthest from the (+) electrode with red wires.
Learn to pipette
simulator
Great Job! Now we are ready to begin pipetting the DNA samples into the wells.
For pipetting tips, you can use our Learning to Pipette Simulator
Next
1. 2. 3. 4. 5.
Plan Your DNA Drawing
DNA Parts
Get your DNA parts and organize them based on size. Remember the smaller the DNA, the quicker it will travel down the gel. For example DNA part 5 will travel further than DNA part 1.
We will take advantage of that to create pixel art with the bands that the DNA will form.
7750 bp 5100 bp 3900 bp 2500 bp 1000bp
Next!
A
B
C
D
E
Think of the gel cast as a grid. Its a good idea to get a paper and pencil to plan out your image and calculate the amounts of DNA parts you will want to add to each well. For every well you will want to dispense around 10 ul of material be it DNA or filler fluid.
In the example above, we’d be placing 2 uL of DNA part 1 and 2 ul of DNA part 5 in well C to create the “eyes” of our smiley face. We’d also add 2 uL of DNA Part 1, Part 2, Part, 3, Part 4 and Part 5 to well E to create the smile
1. 2. 3. 4. 5.
Wells
0 ul
C
D
You’ve already added 10 ul to that well!
Click to select your DNA part. Then, click on a well to pipette that DNA part into it. Each click will be 2 uL of DNA
Restart
E
A
B
Put the electrophoresis tank lid on, making sure the red wire and black wire are in their correct positions.
Set it to a voltage between 80-150 volts. Generally, a good spot is around 100 V for agarose gels. If you go too high the gel will heat up too much and your samples will look much more blurry than if you run at a lower voltage.
Turn on power supply
10 ul
You need to add enough filler fluid to have a total of 10 uL in each well.
Add the filler and run the gel!
Think of the gel cast as a grid. Its a good idea to get a paper and pencil to plan out your image and calculate the amounts of DNA parts you will want to add to each well. For every well you will want to dispense around 10 ul of material be it DNA or filler fluid.
In the example above, we’d be placing 2 ul of DNA part 2 and 2 ul of DNA part 5 with 6 ul of filler for a total of 10 ul.
Why is it a good idea to plan?
In general you don’t want to wait more than 10 minutes from the start of loading your samples to starting to run your gel. This is because while the samples fell to the bottom of the well when you initially loaded them, they will slowly mix with the TAE buffer in the tank and can start to diffuse out.
Keep the tank stationary. If the 1x TAE buffer moves back and forth over the wells, it will start mixing with your samples and they will leave the wells to mix into the tank. Your samples would be lost!
Got it!
Lets watch a timelapse under a blacklight!
You will run the gel at 100 V for 30-60 minutes. You’ll see that the blue/purple dye in the DNA parts will run quite fast. The dye is of the right charge and size to run at the leading edge, a little bit faster than the smallest DNA Part. When the blue/purple dye reaches 1 cm from the bottom of the gel you can stop running the gel.
10000 bp 7500 bp 5000 bp 4000 bp 3000 bp 1500 bp 750 bp 400 bp
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Did your drawing turn out like you planned?
You can use the button go back and try again
Try it again!
DNA Ladder
Add the DNA ladder
If you add your DNA sample to a well and run it with a ladder, you can estimate your DNA fragment size by comparing it to the ladder, such as in the following example.
Or learn more: Analyzing DNA Bands with FIJI Youtube video
Paint with Colourful Bacteria!
Learn to pipette
Try the kits!
Learn Bioplastics
You’ve completed the Gel electrophoresis Simulator!
What is DNA?
Back to Start
Balance a
centrifuge
Genetic
engineering
Check out our other online simulators:
Congratulations!
What is DNA?
Learn Bioplastics
Paint with Colorful bacteria
Genetic Engineering
Balance a centrifuge
Try the kits!
Pipetting Tips!
To add the samples, you want to have the tip of the pipette just barely in the well so that as your dispense the liquid will gently fall down into the well. This is the most difficult part of gel electrophoresis, but you can do it! It simply requires some practice
Ask a friend to stand perpendicular to the wells so they can guide you as you insert the pipette tip in the well. An extra set of eyes can help out a lot! It can help you get context during your first attempts at loading a gel.
Amino Labs Website
Great Job! Now we are ready to begin pipetting the DNA samples into the wells.
For pipetting tips, check out Amino labs online resources or Pipette-it Kit to get started!
When this step is complete you should see small indentations in the gel, called wells. These are where your samples will be loaded.
Great Job!
Continue to learn how to pipette your DNA into the gel, how the DNA will “run” inside the gel, and practice your new skills.
You’ve learn to make an agarose gel and get set up to do gel electrophoresis.
Once your cast and comb are assembled and you’ve placed them on a flat and stable surface, you can move on to the next step.
Lets learn how to pipette DNA into the wells in our gel.
Insert the pipette into the gel, and lower the pipette to the right depth, about 2-3 mm into the gel
Be careful not to jab the bottom of your gel!
Hold to lower the pipette
You went too far down and jabbed into the gel! Try again.
Now that your DNA is in the well, you need to add your DNA ladder. What is a ladder?
The DNA ladder contains DNA fragments of known lengths. When you run a DNA ladder in an agarose gel, the DNA parts will separate by size. It is similar to a ruler you would use to measure paper or wood
Lower the pipette
Pipette the ladder just like how you did for the DNA in the well!
Watch the Timelapse of the DNA ladder!
The smaller DNA travels furthest in the gel.
10000 bp 7500 bp 5000 bp 4000 bp 3000 bp 1500 bp 750 bp 400 bp
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Using a simulation of AminoLabs: Gel Electrophoresis Kit: Draw with DNA you will use different sizes of DNA parts to create a pattern in the gel.
You know how to make and run gels!