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Building and Running a Homemade Agarose Gel Electrophoresis by

Caylee_Collins (/member/Caylee_Collins/) in science (/explore/category/technology/channel/science/)

Download h (/id/Building-and-Running-a-Homemade-Agarose-Gel-Electr/) 19 Steps .

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About This Instructable

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Sep 13, 2015
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(/id/Building-
Introduction and-
Running-a-
A gel electrophoresis is a tool utilized by molecular geneticists to separate and Homemade-
view different parts of macromolecules such as DNA, RNA, or proteins. This Agarose-Gel-
technique works because most macromolecules are negatively charged. When Electr/)
they are placed in the gel wells on the negative end of the chamber and an
electric field is applied, the molecules will migrate to the positive end through the Add instructable to:
gel’s microscopic holes. The smaller and more negative the molecules are, the ; Group
further they will migrate to the positive end. This results in separation of the
macromolecules based on mass and charge. If the molecules are dyed, a
person can observe the distinct bands and interpret the results. The results of a
Related
gel electrophoresis have many applications, including forensic investigations,
paternity tests, and measuring protein levels. DIY Simple & Cheap
Electrophoresis Setup for
This Instructable explains all the steps necessary to gather the necessary DNA Separation (/id/DIY-
materials and tools, construct your own gel chamber and comb, make a 1% Simple-Cheap-
(/id/DIY- Electrophoresis-Setup-for-
buffer solution, make a1% Agarose gel, and run the gel electrophoresis with Blue LED transilluminator
different colors of food dye. This will allow you to separate and compare the (/id/Blue-LED-
molecules in three colors of food dye. transilluminator/)
by jorodeo (/member/jorodeo/)
This experiment is recommend for people of at least high school age wanting to (/id/Blue-
DIY Gel Electrophoresis Box
learn about the principles behind this molecular genetics technique through for Separating DNA (/id/DIY-
actively experimenting. Knowledge of molecular genetics or the associated lab Gel-Electrophoresis-Box-for-
processes are not required to conduct this experiment. Separating-DNA/)
(/id/DIY-Gel- by abizar (/member/abizar/)
The end of this document also contains a section to aid you in interpreting your UV Transilluminator (/id/UV-
Transilluminator/)
results. A troubleshooting section is also provided to help you have a successful by jorodeo (/member/jorodeo/)
experiment.
 Safety Hazards How to Prepare an
Electrophoresis Argarose
None of the materials required for this experiment present any significant Gel (/id/How-to-Prepare-an-
Electrophoresis-Argarose-
chemical or health hazard. While the Agarose powder and/ or gel is not toxic, be
(/id/How-to- Gel/)
sure NOT to ingest it. Also, show caution when setting up and running the
battery-powered electric current. Care should also be taken when handling the
heated Agarose gel. Be sure to wear oven mitts when handling the heated
Agarose gel.

Step 1: Materials/Equipment List and Cost/Time

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Materials/Equipment List
Materials

Small, rectangular plastic box –A travel soap box will work well.

Stainless Steel Wire – 20 Gauge A Piece of Foam or Cardboard – At least 4 by

4 inches.
5– 9 Volt Batteries

Agar Powder – 1/4 teaspoon (1 gram)

Baking Soda– 1/2 teaspoon (2 grams)

Distilled Water– 200mL

2 Alligator Clips with Leads
 Food Coloring Dyes – Red, Blue, and Green
Tools

Wire Cutters

Scissors

Measuring Spoons – Size: 1/4 and 1/2 teaspoons

Small Mixing Bowl

Microwave-Safe Bowl

Heat-Resistant Spoon

Microwave
Plastic Syringe (mL)

Butter Knife Ruler (cm)

Cost and Time

One run of this experiment may cost you between $50 to $75. This price range
is subject to vary depending on how many items you do not currently posses.
This prices range does not include common house hold items: microwaves,
bowls, oven mitts, rulers, scissors, wire cutters, pencils, butter knifes, and
spoons. This experiment should take approximately 1 hour and 30 minutes.

Purchasing Notes

All of these materials and tools can either be found at Walmart and/or
Amazon.com. If you are wishing to run the experiment multiple times, you will
need to purchase more batteries, since they will be drained fairly quickly. Five
fresh batteries are needed each time the experiment is ran. Some sellers of
Agar Powder could require you to be associated with a school.

Step 2: Procedure: Part 1: Gel Electrophoresis Chamber

Preparation: Step 1

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1. With your wire cutters, cut out two equal pieces of the stainless steel wire.

Both of these pieces should be about 2 cm longer than the width of your
rectangular, plastic box.
 This box will serve as the gel chamber. The wires need to be longer than the
width of the gel chamber so they can still span across its width after the next
step has been completed.

Step 3: Procedure: Part 1: Gel Electrophoresis Chamber

Preparation: Step 2

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2. Bend the extra 2 cm of wire into a hook.

This will allow the wire to hook over the side of the chamber. Please see the
Figure #2 as an example.

Step 4: Procedure: Part 1: Gel Electrophoresis Chamber

Preparation: Step 3

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3. Place one wire on one end of the chamber, and the second wire at the other
end.

Please see Figure #3 as an example.These wires will serves as the positive and
negative electrodes, respectively.
Step 5: Procedure: Part 1: Gel Electrophoresis Chamber
Preparation: Step 4

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4. Assemble the 5 9-Volt Batteries.

Please see Figure #4 as a guide on how to assemble the batteries. A positive

terminal of one battery will be snapped into a negative terminal of another
battery. You need to continue this process with all the batteries until one positive
and one negative terminal is not snapped to another battery.

Step 6: Procedure: Part 1: Gel Electrophoresis Chamber

Preparation: Step 5

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 5. With your pencil, trace out the gel comb from either craft foam or cardboard.
Then cut it out with scissors. Please see Figure #5 as an example of tracing the
comb.

Please use Figure #6 as a reference to how the comb should look.

The comb will need to be wider at the top so it can rest on the edges of the
chamber. Slits can be cut into the outer edges of the comb. This will allow it to
stay upright easier.

The bottom of the comb needs to have three teeth for each color of food dye.

Each tooth should not be touching the bottom of the gel chamber. There needs
to be around 1/2 cm of clearance between the bottom of the teeth and the
chamber.

The teeth also need to be evenly spaced away from each other. Please see
Figure #7 as an example.

The size and spacing of your teeth will depend on the dimensions of your
chamber. A 10cm by 6 1/2 cm chamber will require at least 1 cm spacing for 3/4
cm teeth.

Step 7: Procedure: Part 2: 1% Buffer Solution Preparation:

Steps 1 and 2

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 1. The buffer solution will need to be a 1% solution of baking soda. Combine 1/2
teaspoon of baking soda with 200 mL of distilled water in your mixing bowl. Use
the plastic syringe to measure out the water.
2. Stir the solution well with a spoon.

Step 8: Procedure: Part 3: 1% Agarose Gel Preparation: Step

1 and 2

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1. Place 1/4 teaspoon of agar powder in the microwave safe bowl.

2. Add 100 mL of the buffer solution made in the previous steps to the agar
powder in the microwave safe bowl.

Step 9: Procedure: Part 3: 1% Agarose Gel Preparation: Steps

3, 4 and 5

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 (/file/F8LQH4DIEGXUIWP/)

3. In order to dissolve the agar powder, place the bowel in the microwave and
turn it on for 1 minute. Do not forget to wear your oven mitts when handling the
heated mixture. (Figure #10)

4. Every 15 seconds, stop the microwave and stir the solution.

Keep doing this process until the solution begins to bubble.

Once this happens, quickly and carefully remove the bowl from the microwave to
avoid having the solution bubble over.

5. Make sure the solution is translucent. If it is not, you will need to remake your
gel. Please reread through the steps to ensure you are following them correctly.

Please see Figure #11 in order to check the color of your gel.

Step 10: Procedure: Part 4: Preparing to Run the Gel

Electrophoresis: Steps 1, 2, and 3

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 (/file/FF5KPJDIEGXUJC0/)

1. Make sure you have made the gel chamber, comb, buffer solution, and agar
gel before moving on to this set of instructions. (Figure #12)

2. Next with the metal electrodes out of the plastic gel chamber, place the comb
into the gel chamber.

3. The comb should be about ½ cm from one of the ends of the gel chamber.
This 1/2 cm will provide room for you to cut a line in the gel in a later step with a
butter knife. (Figure #13)

Step 11: Procedure: Part 4: Preparing to Run the Gel

Electrophoresis: Steps 4 and 5

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4. Now it is time to carefully and slowly pour the 1% Agarose gel into the gel
chamber. Stop pouring when the Agarose gel has covered approximately 1/2 cm
of the comb’s teeth. (Figure #14)

Tip: You can utilize the plastic syringe to move the Agarose gel into the
chamber. Be sure to rinse the syringe out with distilled water after you have
finished this.

Depending on the size of your chamber, you may not need to pour all of the
Agarose gel.
 5. Place the gel chamber in a safe place and wait at least 30 minutes for the
Agarose gel to solidify. The gel will have the consistency of jello when it has
reached room temperature and is ready. Do NOT proceed on to the following
steps until you are sure the gel has completely cooled!

Step 12: Procedure: Part 4: Preparing to Run the Gel

Electrophoresis: Step 6

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6. Gently pour the rest of the buffer solution over the top of the gel in the
chamber. The buffer solution should completely cover and submerge the gel.
(Figure #15)

Depending on the size of your chamber, you may not need to pour all of the
remaining buffer solution.

Step 13: Procedure: Part 4: Preparing to Run the Gel

Electrophoresis: Step 7

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7. Next, firmly grab onto the top of the gel comb and carefully pull it straight up
and out of the gel. (Figure # 16)
 Be extra careful with this step. The wells formed by the comb in the gel will hold
the food coloring for this experiment.

Step 14: Procedure: Part 4: Preparing to Run the Gel

Electrophoresis: Steps 8 and 9

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8. You will need to make room for the wire electrodes. Take a butter knife and
cut 2 lines across the width of the chamber on both ends. You can cut all the
way down to the bottom of the chamber.

Please see Figure #17 below as a guide.

9.Then, place the wire electrodes back in the chamber. The electrodes should
have the same placement as before. Refer back to Figure #3 above for a
reminder of the electrode placement.

It is highly important the entire length of the electrodes are under the surface of
the buffer solution. The hook of the electrode does not have to be submerged
though.

Step 15: Procedure: Part 5: Running the Gel Electrophoresis:

Steps 1, 2, 3, 4, 5 and 6
 (/file/FD72V3DIEIUBR4R/)

1. One at a time, each of the three different colors of food coloring will need to
be placed in its own well with the syringe. Only a couple drops of each dye are
needed

The less dye placed in the well, the clearer the results of the experiment will be
in the end.

Tip: Place the tip of the syringe loaded with food coloring beneath the surface of
the buffer solution and slightly inside the well before you push the plunger to
release the dye.

Figure # 20 above depicts the technique of loading food coloring into a well.
2. First, fill the plastic syringe with red dye. Deposit the red dye into the well
farthest away from you.
3. Rinse out the plastic syringe.

4. Next, fill the plastic syringe with blue dye. Deposit the blue dye into the middle
well.
5. Rinse out the plastic syringe.
6. Finally, fill the plastic syringe with green dye. Deposit the green dye into the
well closet to you.

Note: The color ordering in Figures #18 and #20 does not match up to the above
schema.

Step 16: Procedure: Part 5: Running the Gel Electrophoresis:

Steps 7, 8, 9
 (/file/FKMU96RIEGXULEN/)

(/file/FZMTQ5YIEGXULFW/)

7. Place one clip of one of the alligator leads on the exposed positive battery
terminal. Place the other end of this same lead onto the positive electrode. The
positive electrode should be the wire farthest away from the wells.
8. Now, take the other alligator clip lead; one of its clips should be clipped onto
the exposed negative battery terminal. Place the other end of this same lead
onto the negative electrode. The negative electrode should be the wire closest
to the wells.

9. At this point, you should be seeing bubbles bubbling up from the electrodes in
the buffer solution because the current is passing through them.
If you do not see bubbles, please see the "Batteries" Section under
“Troubleshooting” in the section below.
Please see Figure #18 and 19 for an example of these steps.

Step 17: Procedure: Part 5: Running the Gel Electrophoresis:

Steps 10 and 11
 (/file/F8IJ6X7IEGXUA88/)

10. The food coloring will migrate from the side of the chamber with the negative
electrode to the end with the positive electrode.
11. Keep running the gel until food coloring bands have clearly and distinctly
separated away from each other. While this process is occurring, be sure to
check it at least every 5 to 10 minutes.
This whole process usually takes around 15 to 30 minutes.

Step 18: Interpreting the Results

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 The secondary color of food dye separated into different colored bands after the
gel electrophoresis ran. This is because the secondary colored dye contained
different sized macromolecules within it. Different sized molecules will separate
at different speeds, resulting in bands at certain locations down the length of the
gel. The same process occurs with DNA and other macromolecules.

Figures # 21 - 23 are sample results of this experiment.

Please note the order of the food dye colors are different from the previous
instructions.

All the well lanes labeled "Green" have separated out into one band of yellow
and one band of blue.

Step 19: Troubleshooting

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Please read this section if you are encountering problems with this experiment.

Gel Comb
If you find that your comb’s material is not forming distinct enough wells, try
using a sturdier material. One example is cardboard, if you are not already using
it.
Agarose Gel
 If your gel appears lumpy, most likely the Agarose powder did not fully dissolve.
If you have not poured the buffer solution on it yet or ran the gel, you can place
the gel back in the microwave safe bowl and re-melt the solution. Once the agar
has fully dissolved, pour it back into the gel chamber to solidify. If you have
already done one of the above tasks, you will need to start over with new agar
and buffer solution.
If your gel is taking over 35 minutes to firm up, the ratio of agar to buffer solution
is probably incorrect. You will need to remake the 1% Agarose gel with new
materials.
Batteries

The most common problems with this experiment center around the batteries. If
your gel is not bubbling when you attach the alligator clips, first check to make
sure all the batteries are firmly and properly attached to one another. You can
also check to see if the alligator leads are firmly attached to the batteries and the
wire electrodes. If this does not solve your problem and you have used a battery
before for a previous device or another gel run, please swap out that battery for
a fresh one. Running the gel electrophoresis will really drain the batteries. If your
solution is still not bubbling, you can try making new electrodes with fresh wire
and using new alligator leads.

Dyes Keep Running Together?

Having a greater amount of space between wells will help keep the dyes from
running together. This principle has already been implemented in your
instructions. If you find that you are still having the dyes run together, try
increasing the space between the gel wells by changing the dimensions of the
teeth in the gel comb and rerunning the experiment. You might also try reducing
the amount of dye you insert into the wells. It is okay if some of the dye comes
up out of the well and slightly mixes with the other two colors nearby; however,
try not to use too much dye.
Did Your Dye Not Run the Correct Way?

You probably got the positive and negative electrodes mixed up! Please review
Figures #18 and 19 to ensure you are running the gel the correct way. Also
make sure you have the alligator leads connected to the right battery terminal so
they match up to their respective electrodes.

Comments

We have a be nice comment policy.

Please be positive and constructive. w I Made it!  Add Images Post Comment

NyQuO (/member/NyQuO/) 2016-01-24 Reply

i noticed the instructable experimented with three dies...what would the results
be if i tried with dna extracted from fruits?

Flag
threeoutside (/member/threeoutside/) 2015-09-14 Reply

Caylee, sorry, I didn't notice that there are two Step 9s - Instructables is
following the current fad of junking up so much screen real estate with header
panels & ads that my field of view is only about 1/4 of my screen regardless
how I shrink or magnify the View, and I find that constantly disorienting. God
help me if I lose my place in a text. The one I meant was Step 16: Procedure:
Part 5: Running the Gel Electrophoresis: Step 9.

Flag

Caylee_Collins (/member/Caylee_Collins/) (author) . threeoutside

(/member/threeoutside/) 2015-09-14 Reply

Dear threeoutside,
Haha! Your comment about Instructables is so funny and true!

Yes, for this Step 9 I do mean the bubbles should be bubbling up in the
buffer solution. The Agarose gel solidified in "Step 11: Procedure: Part 4:
Preparing to Run the Gel Electrophoresis: Step 5." The buffer solution
was added in "Step 12: Procedure: Part 4: Preparing to Run the Gel
Electrophoresis: Step 6." After the electrodes are put in place in the
Agarose gel and the alligator leads attached, the bubbles will come off of
the electrodes and bubble up through the buffer solution. If you think it
would make the instructions clearer I can change this line in your Step 9
"bubbling up from the electrodes in the buffer solution" to "bubbling up
from the electrodes THROUGH the buffer solution".
Thanks for your comment!

Flag

threeoutside (/member/threeoutside/) . Caylee_Collins (/member/Caylee_Collins/)

2015-09-14 Reply
Aha! No - it was my own fault for not reading carefully. I
had forgotten there's a liquid layer of buffer solution. It's been oh, 30-some
years. Egads.

Flag

threeoutside (/member/threeoutside/) 2015-09-14 Reply

This is really cool. I always enjoyed doing electrophoresis in the research labs I
worked in years ago, and now I can't wait for my granddaughter to get old
enough to try this. One question: In Step 9, do you mean "gel" instead of "buffer
solution"? It tripped me up because I couldn't see where anything was in a
buffer solution at that point.

Flag

Caylee_Collins (/member/Caylee_Collins/) (author) . threeoutside

(/member/threeoutside/) 2015-09-14 Reply

Dear threeoutside,

Are you referring to "Step 9: Procedure: Part 3: 1% Agarose Gel

Preparation: Steps 3, 4, and 5"? Or, do you mean the Step 9 in "Step 16:
Procedure: Part 5: Running the Gel Electrophoresisis: Steps 7,8, 9"?
Sorry for the confusion; Instructables automatically proceeded my titles
with "Step X" and then my chosen title.

Flag
 baecker03 (/member/baecker03/) 2015-09-13 Reply

assuming that the experiment relies on a voltage present. current limiting power
supplies reaching 45 volts can be had and would eliminate the need for
extensive 9 volt batteries.

Flag

baecker03 (/member/baecker03/) . baecker03 (/member/baecker03/) Reply

2015-09-13
expensive*

Flag

DIY Hacks and How Tos (/member/DIY+Hacks+and+How+Tos/) 2015-09-13 Reply

Cool project

Flag

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