Lab 4: Gel Electrophoresis
Agarose Gel:
After PCR amplification of a gene, it is important to determine whether or not the
reaction worked properly. For this, a 1% agarose gel is used. A portion of the reaction
sample is applied to a sample well in the agarose gel and by running a current through the
gel, the DNA (which is negatively charged) starts to move towards the cathode in the
circuit. Since the DNA must travel through the 1% agarose, it moves at a rate
proportional to its size, that is to say larger pieces of DNA move slower than small pieces
of DNA. To determine the size of the DNA in the sample, a standard known as a DNA
ladder is applied to a well adjacent to the sample well. The DNA ladder consists of DNA
fragments of known and varying lengths that can be used to compare with the unknown
sample. See Figure:
To “see” the sample, the agarose gel also contains Ethidium Bromide, which
binds to DNA. Under UV light, the Ethidium Bromide and DNA complex “glows” and
the sample can be photographed.
Wells
DNA ladder
Samples
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Pre-lab Exercise 4:
1. In an agarose gel, does DNA move towards the positive or negative
electrode(1 point)? Why (1point)?
2. What is the role of Ethidium Bromide in the agarose gel (2 points)?
3. What is the DNA ladder (2 points)?
4. How does the agarose gel separate different length fragments of DNA (2
points)?
5. Is it safe to hold/touch the agarose gel without gloves (1 point)? Why or
why not (1 point)?
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Laboratory Exercise 4:
Supplies (per person unless otherwise stated):
 Bunsen burner
 Ethanol spray bottle
 Paper towels
 Kim wipes
 Flint
 Waste container
 Laboratory tape
 Laboratory marker
 Ethanol resistant marker
 Bucket of ice
 Pippettes, 0.5-10 l, 5-50l, 20-200l, 100-1000l (1 set per 2 persons)
 Pippette tips for all volumes
 Agarose gel for each student
 Gel box
 Gel power source
 Small Tupperware tray
 Gel imaging system with printer and disk drive
 Saran wrap
 Parafilm
Solutions:
 PCR samples
 DNA samples
 Loading dye
 1 kb ladder
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Gel Electrophoresis Protocol
___ 1. Remove your PCR samples from the refrigerator.
___ 2. Cut a strip of parafilm about 4 inches long by 1 inch wide.
___ 3. Add 1 l of loading dye to 10 l of your sample on the strip of parafilm
(use the parafilm as a surface to do your mixing)
___ 4. Load the now blue colored sample deep inside the wells of your gel.
One sample per well. Make certain to leave at least on lane open for the
DNA ladder (your reference).
___ 5. If you have many empty wells, please load 10 l of your extracted DNA
sample along with your PCR samples. (This will help with troubleshooting in the event that the PCR failed. One of the first things we’d
check would be our DNA.)
___ 6. Make certain your electrodes are placed such that your sample is running
from the negative side to the positive.
___ 7. Run the gel at 100 Volts.
___ 8. Once finished running, gently remove your gel from the tray, place in a
carrying tray and photograph your gel.
___ 9. Look for the 1.5kb products!
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Report Guidelines for Laboratory 3 & 4:
1. When pipetting small volumes, why do we pipette directly into liquids rather than
the walls of tubes (1 point)?
2. Why did we prepare for 7 reactions when we only have 4 samples (1 point)?
3. What primers where used in the PCR and what size product is expected (2
points)?
4. What is the concentration of agarose in your gel? What would you expect if you
increased this concentration (2 points)?
5. Attach your gel image in the space below (2 point):
6. Label all your wells (directly on gel photo) and describe the contents of each well
(4 points)?
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7. Mark a line along the gel photo where you expect your product. Do you have any
product in your wells? Describe your results for each of your samples (2 points).
8. Did your positive/negative controls behave properly (1 point)?
9. If any of your samples did not amplify, can you identify possible sources of error
in your experiment. In other words, did anything go wrong during the experiment
that might result in failed reactions (1 point)?
10. Assume none of your soil samples amplified but your positive control did. What
can you conclude from this (1 point)?
11. Assume some of your soil samples amplified but your negative control did also.
What can you conclude from this (1 point)?
12. Assume all your reactions (soil samples and positive control) failed. What can
you conclude from this (1 point)?
13. If you were able to run your DNA directly on the gel, describe those results. Did
you successfully extract DNA from soil (1 point)?
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14. What conclusions can your reach when comparing your PCR results with the
color of your DNA extract (1 point)?
15. If troubleshooting, what might your try next (2 point)?
16. When presenting data at a scientific conference, you must describe the methods
you used to your audience. Summarize your procedures thus far (all labs and
field trip). Be Brief. (2 points).
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