1. Electrophoresis
The purpose of electrophoresis is to separate molecules
of DNA, RNA or protein.
Separation can be based upon:
Size
Shape
Isoelectric point (protein only).
Separation is achieved by using a matrix (gel) and an electromotive
force to propel the molecules through the gel .
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2. Setting Up an Electrophoresis Gel
To run a gel electrophoresis you will need
Gel tray
Gel comb
Electrophoretic gel box
Agarose powder
Buffer solution
Dye
Electric field
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3. Electrophoresis involves:
1. Preparation of the gel tray
2. Melting the agarose in a buffer solution
Addition of dye to the molten agarose solution (optional)
4. Pouring the molten agarose into the casting tray with a comb
5. Letting the gel solidify
6. Submerging the solid gel in buffer solution in the gel box
7. Removing the comb to reveal the wells in the gel
8. Loading the samples into the wells
9. Applying the electric current and “running” the gel
10. Analyzing the samples separated in the gel
great demo
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4. Determining the size of gel needed
One must first decide what size gel will be run. In general:
For a small gel tray, about 50 ml of gel is needed
For a medium gel tray, about 100 ml of gel is needed
For a large gel tray, about 200 ml of gel is needed
(note: these are estimates, the volumes will vary with the apparatus used!
Pictures of different size gel trays
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small
medium
large

5. Preparing Your Gel Tray
To form a mold for the hot liquid gel, the tray must be taped on the open sides
To accomplish this pull one piece of masking tape over the open side leaving about 1 cm on the bottom to fold it over thus making a good seal
Pour the gel in and insert the gel comb
Let the gel cool
Remove the tape and the comb and the gel is ready for use
I think I need a camera and I will take some pictures to include in this section
Note: some electrophoresis apparatuses come with gel-casting boxes
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6. Making the Gel Solution
Weigh out the appropriate amount of agarose powder
For example 100ml of a 1.6% Agarose solution will need 1.6 grams of agarose
Pour the agarose powder into a Erlenmeyer flask (the flask should be at least 2X the volume needed to avoid boiling over when the solution is heated)
For example 100 ml of gel solution will be made in a 250 ml Erlenmeyer flask
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7. Making the Gel Solution
Agarose Powder
This is commercially available
Has the consistency of fine sugar
Non-toxic
Weighing out the powder
Use a weigh boat and scale
Tare the scale with the empty container
Add an appropriate amount of agarose
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8. Making the Gel Solution
Add the appropriate volume of buffer to the agarose powder
Commonly TAE or TBE are used for buffers.
The buffer controls the pH of the solution and the salts in the buffer improve conduction of electricity
TAE and TBE buffers are available commercially
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9. Making the Gel Solution
Cover to prevent water loss and microwave initially at high power for 60 seconds per 100 ml of solution
Remove and carefully stir by swishing the solution (the solution may be superheated so wear oven gloves!)
Return solution to the microwave and keep heating and swishing for 30 second intervals until all the agarose powder has dissolved
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Note: this can also be done using a hot-plate!

10. Pouring the Gel
After the gel has cooled (to 60C) and before it starts setting, the gel solution must be poured into the prepared gel tray
Pour slowly to ensure no bubbles form
Pour until the comb teeth are at least 50% covered by the gel
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11. Removing Bubbles that Form
If a few bubbles form, use a disposable pipette tip to move the bubbles to one side of the gel tray – keep the bubbles away from the middle of the gel and the comb.
Bubbles can interfere with DNA progressing evenly through the gel
Let the gel set until it become opaque
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12. Placing the Gel Tray into the Tray Box
Carefully remove the tape from your gel tray
Place the gel tray (with gel) in the tray box
Add the Tris buffer (same buffer that was used to form the gel) into the tray box until the level of buffer is 4 or 5 mm above the gel
Remove the comb carefully- the holes made by the comb will become the wells. This is where the DNA samples are placed
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13. Remove the tape carefully from each end of the gel
Carefully remove the comb.
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14. Preparing the DNA Samples
To properly run a gel, one must add a loading buffer to the samples before they are placed into the wells. The purpose of the loading buffer is three-fold
To track the progress of the electrophoresis
To make it easier to load the wells
Add to the density of the DNA sample so the solution will sit in the well
Decide on the sample order (and take note of this in lab notebook!)
Put up to 20 µl of a DNA sample in a clean tube
Add 5 µl of loading buffer to the sample
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15. Loading Your Gel (Part 1)
Place a piece of black paper under the tray box to help visualizing the wells.
Carefully withdraw the sample from the tube with a pipettor.
Gently pipette the sample into the well
Keep track of the order in a notebook!
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16. Loading Your Gel (Part 2)
Remember to leave some wells free for the ladders
Ladders are size standards that assist in identifying the size of a sample after the gel has run
You should leave a space between your samples and the ladders
The last wells on either side of the gel are usually left empty as these lanes do not run properly
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17. Attaching Your Tray Box to a Power Supply: Part 1
Place the cover on the tray box and attach the electrodes
Remember “DNA runs to Red”
The electrode near the wells attaches to the negative terminal (cathode) and the other electrode attaches to the positive terminal (anode).
Once the wires are attached, turn on the voltage.
As a general rule: 5 Volts per cm (distance between electrodes on the tank)
Distance between electrodes
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18. Attaching your Tank to a Power Supply: Part 2
In general up to 100 Volts is used for a small gel and up to 200 Volts for a medium gel
If current is running from the cathode to the anode, tiny bubbles can be seen rising through the buffer solution from both the cathode and anode.
Never stick fingers into gel unless power source is off! Electrocution hazard!!
Run the gel until the loading dye has run at least halfway down the gel for good resolution of the samples
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19. Analyzing Your Gel Ladder Ladder DNA samples Turn off the power supply
Disconnect the wires
Remove the gel tray with the gel from the tray box
Stain gel with a dye that will bind to DNA (we recommend using Fast Blast DNA stain from Biorad)
Size DNA fragments using your DNA ladder
DNA samples
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20. Where to Get More Information
Electronic sources
Dolan DNA Learning Center Electrophoresis Animation
Colorado State: Agarose Gel Electrophoresis of DNA
Mama Ji's Molecular Kitchen
Molecular Biology Cyberlab
Genetic Science Learning Center
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