Name _____________________________

Date __________________

Gel Electrophoresis Lab Questions

Micropipettes: Watch the “Using a Micropipette” video posted on Google Classroom and
answer the questions below.
1. What do the numbers mean in the micropipette names? (i.e. P2, P20, P200, P1000)
a. The maximum volume of liquid each micropipette can hold. For example,
the P1000 can pipette a maximum of 1000 microliters
2. Fill out the table for the range of volumes in microliters (µl) that each pipette can hold.
The first one has been done for you.

Micropipette Volume Ranges

Micropipette Minimum Volume (µl) Maximum Volume (µl)

P2 0.2 2
P20 2 20
P200 20 200
P1000 100 1000

3. What can happen to the micropipette if you measure volumes outside of the appropriate
range?
a. Inaccurate volume measurements
b. Damage to internal mechanisms of the pipette
4. If you have the number 100 listed on the volume indicator (see
image on right) of a P2 and P200 micropipette, are you transferring
the same amount of volume of that sample? Please explain.
a. No, since the decimal place changes. This would be 1
microliter (µl) for the P2, compared to 100 microliters of
the P200 (µl).
5. List the steps of transferring liquids with a micropipette. Be sure to
include all steps involved in 1) setting the volume, 2) attaching
pipette tips, and 3) pipetting volumes. Use key words in the image
below.
a. Setting the volume

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i. Rotate the thumb wheel or the push
button to adjust the number dials
ii. Make sure you adjust volume based
on the micropipette used (e.g. P200
vs P1000)
b. Attaching pipette tips
i. Attach the appropriate pipette tip to
the end of the pipette. P2, P20, and
P200 use the yellow tips, while
P1000 uses the blue tips
ii. Hold the pipette with one hand and
the narrow part in the palm of your
hand
iii. Add pipette tip by gently but firmly
pushing the pipette into the pipette
tip IN THE TIP BOX
c. Pipetting volumes
i. Push the push-button down to the
first top
1. Gently apply pressure to the
push-button with your thumb
until you feel a natural stop
(first stop)
ii. Keep the push-button at this level, place the pipette tip at about 2
mm into the liquid
iii. Release the push-button by slowly allowing the button to return to
its initial position
iv. Pause for a second to make sure all required volume of liquid is
taken up into the tip
v. Withdraw pipette tip from liquid and place in recipient container
vi. Slowly push down the push-button to release liquid in tip to tube
vii. Push beyond the first stop, which makes sure all residual liquid is
expelled from the tip
viii. Touch tip to inside wall of container while expelling the liquid
ix. Fully withdraw pipette from liquid before releasing the push-button
x. Use tip ejector button to release the tip from the pipette
xi. Dispose of the tip in appropriate waste container(s)
6. Please explain the meaning of this image (on the
right) when pipetting volumes. Be sure to include
in your description what is happening in arrows A,
B, C, D, and E and whether the pipette is in the
liquid or not.
a. The image explains the importance of
the first stop when taking up liquid into the tip of the pipette.
A = First, push the push-button (outside of the liquid) to the first stop.

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B = Next, take up the liquid by slowly releasing the push-button while
submerged in the liquid.
C = Then put the pipette in the container you want your sample to go into
(recipient container), pushing to the first stop.
D = While still in the recipient container, gently push the push-button to the
second stop to fully expel all the liquid. Before releasing the push-button,
make sure the pipette is outside of the liquid.
E = Outside of the liquid, slowly release the push-button.
7. What are some common mistakes to avoid when using a micropipette? List at least three
mistakes and the damage they can cause to the equipment or sample.
a. Using a micropipette without a tip  causes the tip to corrode and
contaminates liquids and experiments
b. Use a pipette past its limits (under or over)  pipetting inaccuracies and
damages the pipette
c. Repeatedly jam a tip to the pipette  damages the pipette
i. Repeat the procedure if the tip doesn’t stay on the pipette
d. Push past the first stop when taking up liquids  takes up excessive
volume than accounted for
e. Quickly let go of the push button when taking up liquids
f. Forget to pause after taking up liquids and releasing the button  not
enough time for correct volume to be taken up and takes up air instead
g. Tip stays below the surface when taking up liquids, so as not to absorb
liquid
h. Do not set pipette down with liquid inside the tip  can take in air and
contaminate the sample
i. Forgetting to touch last drop(s) to the side of the tube to fully expel liquids
 loses some (or all) volume of sample

Virtual (Online) Lab: Click the link to go to the website on Gel Electrophoresis and answer the
questions below as you work through the virtual lab.
1. What does gel electrophoresis do to DNA? What else can it be used for?
1. Sorts and measures DNA strands according to length.
2. How does DNA move through the gel? How does it use electricity?
1. The DNA moves through the holes in the gel
2. The electric current helps DNA move through the gel
3. What size molecules move quicker?
1. Smaller molecules
4. What is stain used for?
1. Stain the sorted groups of DNA to make large groups of stained DNA
strands visible. These are the bands in the gel
5. Describe the procedures (and materials) involved in running a gel electrophoresis for
each of the five steps below. Draw a picture for each step that summarizes important
procedures in that step.

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Step 1: Make the gel
Materials: powdered agarose, buffer, flask, microwave, gel mold, gel comb
1. Put small amount of agarose into the flask
2. Add liquid buffer to the flask
3. Heat the mixture in the microwave until the agarose melts in the buffer
4. Pour the melted agarose mixture into the mold (that has tape on each end
to hold the liquid)
5. Place the comb into the gel
6. Let the gel cool and solidify
7. Remove the comb
Step 2: Set up the gel apparatus
Materials: gel, electrophoresis box, bottle of buffer
1. Pour buffer into electrophoresis box
2. Place gel (in mold, but without the tape on ends) into the electrophoresis
box
i. Gel should be just barely submerged in the buffer
Step 3: Load the DNA sample into the gel
Materials: loading buffer, tube of DNA, DNA size standard (ladder),
micropipette, electrophoresis box with buffer and gel, and pipette tips
1. Suck up loading buffer with the micropipette, then add to DNA sample
i. Loading buffer dyes DNA sample for better visibility and makes
sample thicker to drop into well
2. Transfer DNA sample from the tube into the well of the gel
3. Transfer DNA size standard (ladder) into well
ii. Gives you a reference for lengths of DNA strands
Step 4: Hook up the electrical current and run the gel (mention which color cord is
closest to the wells and whether this is a positive or negative charge)
1. Plug the black cord from the electrophoresis box in to the power supply
i. This is a negatively-charged cord that is closest to the wells
2. Plug the red, positively-charged cord in to the power supply
3. Turn on the power supply
4. Look at the gel box and check for tiny air bubbles coming out of the
electrodes that show a current is running
Step 5: Stain the gel and analyze the results
1. Take mold out of gel
2. Put mold into DNA staining solution. Leave for about a half hour
i. Make sure to wear gloves and other PPE to avoid contact with the
staining solution
3. Remove gel from staining solution and place on UV (ultraviolet) light box
4. Turn on UV box
i. Wear protective goggles to prevent damage from UV light in eyes

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5. Compare bands of DNA sample with DNA size standard (or ladder)
6. Measure base pairs for each band in DNA sample

6. DNA moves from the ___positive___ (positive or negative) end to the __negative___
(positive or negative) end. This happens because DNA is repelled by the __negative__
(positive or negative) charge.
7. __Shorter__ (Shorter or Longer) DNA strands will migrate farther from the starting point
than the __longer__ (shorter or longer) strands.

8. Use the image of the gel electrophoresis bands above to match each lane with the
statement that best describes it. Write the lane number next to the statement. (A lane is
a corridor through which DNA passes as it leaves a well.)
a. This lane contains the shortest DNA fragment 2
b. This lane contains the longest DNA fragment 1
c. This lane contains a 150 base pair (bp) DNA fragment 3

Gel Electrophoresis: Answer the questions below as you work on your gel electrophoresis lab
during class.
1. Draw a detailed sketch of the gel. Use colored pencils to show the colors of the samples
as originally loaded into each well. Draw the different color bands that resulted from
electrophoresis of each dye. Special attention should be paid to the differences in dye
migration and the relative positions of the bands in each lane.

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2. In the table below, record the number of dye bands in each lane and the direction of
migration (positive or negative) for each band. Determine the migration distance of each
dye in the known and unknown samples by measuring the distance from the center of
the starting well to the center of the dye band with a ruler. If the dye moved toward the
positive pole, mark the migration distance as a positive number. If the dye moved toward
the negative pole, mark the migration distance as a negative number.

Number Direction of Migration Migration Distance

Lane Sample
of Bands (positive or negative) (cm)
1 Bromphenol blue
2 Methyl orange
3 Ponceau G
4 Xylene cyanol
5 Pyronin Y
6 Unknown #1
7 Unknown #2
8 Unknown #3

3. What are the differences in the way this gel electrophoresis was done compared to the
DNA virtual lab gel electrophoresis?
a. The wells were in the middle for this lab, while the DNA lab had wells on the
negative side.
b. The molecules ran both sides on this lab, while the molecules only ran on
one end in the DNA lab
4. Were any of the colors pure? Did the different dye pigments migrate different distances?
a.
5. What are the main factors that help separate the dye pigments (list 3)?
a. Electrical current – charged molecules loaded in a gel move into the gel
through an electrical attraction to the oppositely charged pole. Without the
electrical current, migration would not be possible
b. Porous matrix of agarose gels – Pores provide a passageway for sample to
move through the gel; if the gel weren’t porous, migration would not be
possible. Pores act like a sieve, which contributes to molecule separation.
Small molecules maneuver more easily through the pores of the gel than
the larger molecules and thus migrate more quickly than the larger, slower
molecules
6. What charge is carried by the dye pigments? How did you determine this?
a. Dyes that migrated to the positive electrode had a net negative charge.
b. Dyes that migrated to the negative electrode had a net positive charge
c. This occurs because opposite charges attract (and similar charges repel)
7. What color molecule is most likely the smallest? How did you determine this?
a.

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8. Compare the dye pigments in the unknown dye mixture to the pigments of the known
colors. How can you use the results from the known colors to determine what is in the
mixed sample? What dye components are in the unknown dye mixtures?
a. Unknown #1: xylene cyanol and ponceau G
b. Unknown #2: methyl orange, bromphenol blue, and ponceau G
c. Unknown #3: xylene cyanol and pyronin Y

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