Electrophoresis Lab – AP Biology
Between the pHschools online lab (same link as the transformation lab) and the background
provided in this packet, make sure you understand the procedures. To prove you did the online
portion, please print practice problem #1 with the results. You should do the whole self-quiz – it
will definitely help you on the test.
Answer the following pre-lab questions on a separate sheet of paper to turn in the day before the
lab:
1) What is the overall purpose of running the gel that you are going to run? (Don't just refer
to what the patient in the scenario wants to know - - describe why gel electrophoresis will
be used to find her answer).
2) Describe in your own words how you calculate the size of a DNA fragment using a
standard curve.
3) How should the wells be oriented in relation to the positive and negative poles and why?
4) What are the two purposes of the loading dye that the DNA is suspended in?
5) How will you know when to stop the gel?
6) Why must you stain the gel? How is probing different from staining?
7) What are you loading into each of the 3 control lanes (not including the standard) and why
are they needed? Be specific.
8) What should you expect the banding patterns on your gel to look like? Sketch a rough
drawing below showing the expected results for the samples loaded into each lane.
Explain why you drew what you did.
Procedure
1) The gels will already be poured and polymerized and sitting in buffer.
2) Put the gel in the gel box and make sure that the gel is submerged in the buffer.
3) Make sure the gel is properly oriented so that the DNA will run toward the positive pole.
4) Load 35-38 μl of each sample into the proper well. Load sample A in lane 1, sample B in
lane 2, sample C in lane 3, sample D in lane 4 and sample E in lane 5.
A = Standard DNA fragments
B = Control DNA sample
C = Patient peripheral blood DNA
D = Patient breast tumor DNA
E = Patient normal breast tissue DNA
5) Snap the cover down onto the electrode terminals (make sure that the negative and
positive indicators on the cover are properly oriented).
6) Plug the black wire into the black input (-) on the power supply. Plug the red wire into
the red input (+) on the power supply.
7) Set the power supply to 70 volts and start the power. The gel should take about 1.5 hours
to run.
8) Check to see that the current is flowing properly by checking for bubbles forming on the
electrodes.
9) Stop the gels when the tracking dye is near the bottom. Do not allow the tracking dye to
run off – you may lose some of your DNA.
10) When completed, turn off the power, unplug the power source, then unplug the leads and
take the lid off.
11) Transfer the gel to a tray for staining and see the staining procedure section.
12) Measure the distances from the well to the top of each band in found in Lane 1(Standard
DNA). Record each distance in Table 1 given below.
13) Follow the directions on the following pages to graph your standard curve. Then,
measure the how far each DNA fragment in Lane 3 (Sample C) migrated and record
those values in Table 2 below. Using your standard curve, figure out the size of each of
these bands of DNA. Compare to the actual values.
Table 1:
Standard DNA
Actual Baser Pairs
Digest
Measured Distance (cm)
23,130
9,416
6,557
4,361
3,000
2,322
2,027
725
570
Table 2:
Samples
Measured Distance (cm)
Band 1
Band 2
Band 3
Digest
Interpolated base pair #
Cancer Genetics Background Information
Many contributory factors have been identified to cause the onset of cancers, including exposure
to certain carcinogens in our diets and environment. Several forms of cancer have familial
predispositions. These cancers appear to be linked to inherited mutations of tumor suppressor
genes, such as p53.
Familial cancers constitute a very small
fraction of the total reported cancers and
they occur in dominant inherited patterns.
Mutations that are directly inherited are
referred to as germline mutations. A second
type of mutation, known as a somatic
mutation, does not have direct genetic links
and is acquired during the life of the
individual. Patterns of typical hereditary and
sporadically acquired nonhereditary
pedigrees appear in Figure 1 to the right.
In a germline with an inherited mutation, a
single somatic mutation within a suppressor
gene will result in the inactivation of both
alleles since one is already inactivated at
birth. By contrast, normal inherited
suppressor genes, that are free of mutations,
will require two sequential mutations to
initiate tumors. This model is referred to as
the "Two-hit" hypothesis.
In recent years, the p53 tumor suppressor protein has become the center of many cancer biology
studies. Because it appears to be of major significance, there is great impetus to study how this
gene functions in normal cells compared to cancer cells. The gene for p53 encodes a 53,000
molecular nuclear phosphoprotein. Wild type (normal) p53 functions as a cell regulator. There is
now well-documented evidence that normal p53 is a transcription factor. Upon introduction of
mutations, p53 loses its ability to bind to DNA. By contrast, p53 that have mutations in specific
hot spots promote uncontrolled cell growth and therefore function as oncogenes. For a tumor
suppressor gene such as p53 to play a role in transformation in cancer, both alleles need to
be altered.
An inherited disease condition Li-Fraumeni syndrome (LFS) is rare. Families that have LiFraumeni syndrome have high rates of many types of cancer that appear early in life. Cells in the
individuals with LFS have a single wild type p53 allele. In a normal p53 gene, there is no
restriction enzyme site, but a mutation at a hot spot site in the p53 gene creates a
palindromic sequence (CAGCTG) that can be recognized by a restriction enzyme. If a linear
DNA molecule that contains a single recognition site is cleaved once, it will generate two
fragments. The size of the fragments produced depends on how far the restriction enzyme sites
are from each other.
Electrophoresis Background Information
Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA
fragments generated by restriction enzymes. The gel consists of microscopic pores that act as a
molecular sieve. Samples of DNA are loaded into wells made in the gel during casting. Direct
current is then applied to separate the DNA fragments. Since DNA has a strong negative charge
near neutral pH, it migrates through the gel towards the positive electrode during electrophoresis.
Linear DNA molecules are separated according to their size. The smaller the linear fragment, the
faster it migrates. After running the gel, you can stain the gel in order to be able to visualize the
fragments, otherwise you cannot see the clear DNA in the gel. If the size of two fragments is
similar or identical, they will migrate together in the gel as a single band or as a doublet. If DNA
is cleaved many times the wide range of fragments produced will appear as a smear after
electrophoresis. Therefore staining will not be an appropriate method to visualize the bands. In
this case, radioactively tagged probes must be used.
In order to probe the gel, first the DNA in the gel must be transferred to special paper. Then
probes (singled stranded DNA about 10-15 nucleotides long that bind to complementary
nucleotide sequences) are added to the paper. Since probes will bind only to specific sequences,
those are the only bands you will be able to see on the special paper.
Scenario
Upon monthly breast self-examination, Valerie Brown, age 36, found a small irregular mass. She
was concerned because she knew that her mother had a mastectomy when she was in her late
thirties. Valerie made an appointment with her physician, who referred her to a specialist at a
local cancer center, where she was diagnosed as having breast cancer. As part of the medical
work-up, the oncologist had inquired about her family history of cancer. Upon consultation with
her mother, Valerie learned that her father and his family appeared to be free of cancer. However,
in Valerie's mother's family, several cases of cancer have occurred. The familial pedigree she
created strongly suggests Li-Fraumeni syndrome. Valerie has five children: Justin (16), Sheila
(14), Robert (10), Angela (8), and Anthony (6), none of whom show any signs of cancer at this
time. She was interested in the p53 diagnostic test to determine if she had inherited mutations that
could have been passed on to her children.
Valerie has provided a sample of blood and breast tumor tissue to conduct DNA analysis for the
p53 gene. Scientists have isolated and amplified the p53 gene from Valerie’s genome using
polymerase chain reaction (PCR). Valerie's DNA was then digested with a restriction enzyme that
recognizes the mutant sequence at the hot spot site.
You have been entrusted with these digested samples and it is up to you to use agarose gel
electrophoresis to separate her DNA samples and analyze the resulting banding patterns. Is
Valerie a carrier for the p53 gene mutation?
Post-Lab Questions
1. Does Valerie carry the p53 gene mutation? Explain how you know by comparing
and contrasting your results for lanes 2-5. Make sure you mention each lane in
your response to receive full credit.
2. Discuss how each of the following factors would affect the results of
electrophoresis:
a. Voltage used
b. Running time
c. Amount of DNA used
d. Reversal of Polarity
3. If two small restriction fragments of nearly the same base pair size appeared as a
single band, even when the sample was run to the very end of the gel, what could
be done to resolve the fragments and why would each work? (Give 2 things)
4. For which fragment sizes was your graph most accurate? Least accurate? What
does this tell you about the resolving ability of agarose gel electrophoresis?
5. What is the source of restriction enzymes? What is their function in nature?
What are recognition sites?
6. What is the function of electricity and the agarose gel electrophoresis?
7. If a restriction enzyme digest resulted in DNA fragments of the sizes below,
sketch the resulting separation by electrophoresis. Show the starting point,
positive and negative electrodes and the resulting bands. (Size of bands in base
pairs = 2500, 4000, 400, 2000)
8. Use your standard curve graph to predict how far a fragment of 8,000 base pairs
would migrate.
9. How can a mutation that alters a recognition site be detected by gel
electrophoresis?
10. Predict the number of DNA fragments and their sizes if Lambda phage DNA were
cleaved simultaneously with the restriction enzymes Hind III and EcoR1. Refer
to the map below.