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Chemistry 315

Experiment 2:
Unknown DNA Mapping by Restriction
Enzyme Digestion and Gel
In this experiment you will digest an unknown, plasmid DNA with restriction enzymes
and map the fragments with DNA electrophoresis.

Required Readings
1. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis;
Thomson: Belmont, 2007; pp 867-879.
2. See also the section on gel electrophoresis in the modern biology textbook of your
choice; iGenetics by Peter J. Russel provides a good overview of electrophoresis and
restriction mapping and is available in the biology library. The page numbers are
significantly different in each edition.

Deoxyribonucleic acid, DNA, is the ion of inheritance, and the ground floor for any
recombinant technology. DNA is a biopolymer composed of four monomers known as
nucleotides. A single nucleotide consists of a five-membered sugar ring (furanose), a
nitrogen-containing base, and a phosphate group. The bases, called nucleosides, are
adenine (A), guanine (G), thymine (T), and cytosine (C). The structures of each of these
nucleotides is shown in Figure 1.

Figure 1: DNA nucleobases. Adenine and guanine feature purine rings, thymine and cytosine feature pyrimidine

These nucleotides are covalently bonded to one another linearly through phosphodiester
bonds, forming single-stranded DNA (ssDNA). The hydroxyl group on the 3 carbon of
one nucleotide is bound to the phosphate on the 5 carbon of the next, and so on. Each of
the four bases has a complementary base to which it bonds in double-stranded DNA
(dsDNA). Adenine and thymine form two hydrogen bonds with one another, while
cytosine and guanine form three hydrogen bonds. When two complementary strands of
DNA come together at a temperature that is less than its melting temperature, the
strands will hydrogen bond, or anneal. For example the strand TATACGGG will anneal
and form a double strand with ATATGCCC.

The two ends of ssDNA are termed the 5 and 3 termini. The 5 terminus has a
phosphate group bound to the 5 carbon of the furanose ring. The 3 terminus has a
hydroxl group at the 3 position of the sugar. When DNA is written out using the single
letter abbreviations, the convention is to write the bases starting from the 5 position.
However, complementary strands are anti-parallel, so in the example above, the first
referenced strand, TATACGGG, is written 5 to 3, but the complementary strand,
ATATGCCC is written 3 to 5. A similar biopolymer, RNA, closely resembles DNA.
The 2 carbon of the furanose in RNA is hydroxylated, and the thymine base is replaced
by uracil (U). A strand of dsDNA is shown in Figure 2.

Figure 2: Watson-Crick base pairing between complementary strands give rise to the DNA double helix.

The genomic DNA in eukaryotes (plants, animals, and fungi) is double-stranded and
linear. In bacteria, the genome is circular. Bacteria carry their own genomic DNA as well
as smaller circular DNAs called plasmids. Circular dsDNA has an additional layer of
structural complexity, known as supercoiling. If you held the end of one piece of rope in
your left hand and twirled it in your right hand, the rope would begin to coil onto itself.
You could preserve this coiling by tying the two ends of the rope together. The
convoluted structure of the rope once it has been twirled, then tied is akin to the
supercoiled structure of the plasmid.

Plasmids have become integral for many molecular biological applications. Plasmids can
be engineered to contain DNA that encodes a protein of interest and forcibly inserted
(transformed) into bacteria to express the protein. This sort of engineering is often done

with restriction endonucleases, which are commonly referred to as restriction enzymes.
Restriction enzymes selectively degrade DNA. They recognize specific (often
palindromic) nucleotide sequences. Then they break the phosphodiester bonds between
certain nucleotides in this sequence. For examples of restriction enzymes and their
recognition sequences, see Table 1.

Table 1: Some common restriction enzymes. The symbol / shows where the enzyme catalyzes the cleavage
of a phosphodiester bond. R is A or G, N is any nucleotide, and Y is C or T.

Restriction enzyme Recognition sequence

Xho 5-C/TCGAG-3

Restriction enzymes are naturally occurring, and they play a significant role in the
defense mechanism of prokaryotes. The nucleotide sequences they recognize are often
unique to invading DNA, and are not found in the host genome. Thus, restriction
enzymes restrict the DNA that can remain in a prokaryote.

In vitro, these enzymes can be used to insert the DNA encoding a selected protein into a
plasmid. If the plasmid has the recognition site for a restriction enzyme and the gene of
interest is engineered to have the same restriction site, both the plasmid and the protein to
be inserted can be digested by the same enzyme. The amounts of the plasmid, the
encoding DNA and the restriction enzyme can be adjusted according to the relative molar
masses of the plasmid and the encoding DNA, as well as the activity of the restriction
enzyme. One unit of a restriction enzyme is defined as the amount of enzyme required to
digest 1 g of DNA at 37 C in 1 h. The digested and free ends of the plasmid and gene
of interest can then be ligated together with another enzyme. These modified plasmids
can then be transformed into prokaryotes (such as E. coli.) to express the protein.

Mg2+ is a crucial cofactor in restriction enzyme catalysis, and is often included in
commercially available restriction enzyme buffers. Ethylenediaminetetraacetic acid
(EDTA) is often used to control the reactivity of restriction enzymes as it forms a
complex with the ions, effectively removing them from the reaction solution. This will
ultimately stop the restriction digest.

Often micrograms or less of DNA is needed to carry out transformations. How can such a
small-scale mixture of DNA from a restriction digest be separated into its components?
By ethidium bromide gel electrophoresis!

In electrophoresis, an external potential is applied to a medium and used to drive ions

across an electric field. Agarose is a convenient electrophoretic medium. When a dilute
solution of agarose is heated, then cooled, a gel forms with a consistency similar to that
of JELLO (a gel made from starch). In the agarose gel, there is enough separation
between polysaccharide chains to allow for the migration of many different ions.
The phosphodiester backbone of DNA is negatively charged, thus it will migrate through
an electric field.

The mobility of a charged analyte in agarose is size-dependent. Longer dsDNA will be

slower to migrate through the agarose matrix than shorter dsDNA. Supercoiled DNA will
migrate at a different rate than dsDNA of the same length. Because of this size-
dependence, a mixture of DNA of various sizes can be separated. To fine-tune this effect,
the concentration of agarose can be adjusted according to the range of sizes of the DNA
samples. More concentrated agarose can lead to a more dense gel, which can allow for a
finer separation between short DNA samples. Less concentrated agarose can be used to
separate longer DNA samples. The mobility of a DNA fragment is linearly related to the
logarithm of its number of base pairs (bp).

= () + (1)

A standard DNA mixture with DNA fragments of known length (typically called
markers or a ladder) can be used to generate a working curve based on the relation
shown in (1). From this curve, the length of unknown fragments can be approximated.

Dyes are used to monitor the progress of DNA through an agarose gel. Bromophenol blue
is often injected as a loading dye. Its bright blue color can easily be seen in real time as it
migrates across the gel to indicate visually how far the DNA has progressed. Similar dyes
of different molecular weights can provide an approximation of when to stop
electrophoresis based on what the estimated size of a sample is. Ethidium bromide is a
dye selective to DNA to visualize the DNA fragments in an agarose gel after
electrophoresis. This molecule intercalates in between the stacked bases of DNA, and its
fluorescence is enhanced in the intercalated state. The molecule can be excited with
ultraviolet light, and fluoresces in the visible region. Bromophenol blue and ethidium
bromide are shown in Figure 3.

Figure 3: DNA stains. Bromophenol blue is used to track the progress of DNA during electrophoresis. Ethidium
is used to visualize the location of a piece of DNA.

Prelab assignment
Answer the following questions in your lab notebook. Do not copy from the lab manual
or work with other students this is considered cheating and will result in a score of zero.
1. Find and print the MSDS for ethidium bromide. What are the hazards associated with
this chemical, and how might you prevent them? What action should be taken should
these come into contact with your body (e.g. your skin/eyes)?
2. What safety precaution, according to the procedure, is unique to this experiment?
3. Often, a 1% solution of agarose in electrophoresis buffer is used to separate short
DNA strands. To separate long DNA strands, should a more concentrated or less
concentrated agarose solution be used?
4. How does ethidium bromide label DNA?
5. Describe in one sentence how the length of a nucleotide fragment is mathematically
related to its mobility.

6. Complete Table 2 (page 9) in your lab notebook. That is, calculate how many ml of
restriction enzyme and diluent you will add to each digest.
7. The restriction sites of a sample plasmid are shown below. If it is digested using
EcoRI, how many fragments will result? If both HindIII and EcoRI are used to digest
the sample, how many fragments will this produce? (2 pts)

8. You run a gel with two lanes: one with a circular, supercoiled piece of DNA and one
with a circular, non-supercoiled piece of DNA. Both have the same number of base
pairs. Which will run through the gel faster, and why?
9. Show, by drawing an arrow, the direction in which DNA will migrate if the positive
and negative electrodes are positioned as shown. Why will it migrate in that

electrophoresis apparatus
micropipets and tips
FOTO-Phoresis UV Transilluminator

Lambda DNA
EcoRI-HF restriction enzyme
HindIII-HF restriction enzyme
New England Biolabs (NEB) Cutsmart Buffer (10x)
NEB diluent buffer B (1x)
NEB diluent buffer C (1x)
Purple Loading Dye
TrackIt 1 Kb Plus DNA Ladder
0.5x TBE electrophoresis buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.1-8.2)

Chemicals for this experiment can be located at the bench designated for this experiment
and/or in the freezer in the side room of the lab. Dispose of all waste in the container
designated for this experiment. Do not pour waste down the sink! CAUTION: ethidium
bromide is not selective in the DNA it intercalates. Wear gloves whenever you handle
solutions or gels containing this compound.

Please notify a TA if any equipment is missing or if the chemicals are running low.

Restriction enzyme digests
1. Turn on the water bath, and ensure the water bath is set to 37 C. If its not, notify
your TA.

2. You will be using small volumes of sensitive materials, so be sure you understand
how to use an Eppendorf micopipet. If you have any questions, ask your TA before
you begin. Use a fresh pipet tip for every solution, and dispose of used pipet tips in
the appropriate container. Clearly label each microcentrifuge tube you use.
3. Remove the Lambda DNA and restriction enzyme solutions from the freezer. The
enzymes and DNA must ALWAYS be kept on ice. Ice can be obtained from Noyes
467. If necessary, reagents can be thawed by holding them between your fingers for a
couple of seconds.

You will be carrying out the following digestions:

Table 2: The final amounts or concentrations of reagents needed to carry out the restriction enzyme digests.
Reagents are listed along the top row; digestions are listed vertically in the leftmost column.

unknown HindIII-HF EcoRI-HF NEB Diluent Buffer C Diluent Buffer B

DNA (20 Units/uL) (20 Units/uL) Cutsmart
Undigested plasmid 5 L ---- ---- 5 L bring total ----
volume to 50 L
HindIII 5 L 40 Units ---- 5 L ---- bring total
volume to 50 L
EcoRI-HF 5 L ---- 40 Units 5 L bring total ----
volume to 50 L
EcoRI-HF + HindIII- 5 L 40 Units 40 Units 5 uL bring total bring total
HF volume to 50 L volume to 50 L

The ratio of materials in each digest will be 40 units of each restriction enzyme, 5 L
(~0.5 g) of plasmid DNA, and 1x NEB Cutsmart Buffer, in a final volume of 50 L.
You will dilute the NEB Cutsmart buffer with the appropriate diluent buffer to achieve
the appropriate concentration. Note that in the double digest, you will use equal parts
diluent buffers B and C to bring the total reaction volume up to 50 L.

Addition order is: Diluent Buffer(s), NEB Cutsmart buffer, DNA, then restriction

These are the ratios recommended by New England Biolabs.

For each solution:
After the appropriate DNA solution, enzyme, and diluting buffer is in the microcentrifuge
tube, close the lid and flick the tube with your fingernail for 30s to mix them thoroughly.
To keep the minicentrifuge balanced while you spin down the contents of the tubes, load
two tubes of the same volume into opposite slots in the centrifuge. Start the centrifuge,
and do a quick (5 s) spindown. Place the tubes into the microcentrifuge tube holder in the
water bath. Digest for 1 h, making sure the bath temperature remains at 37 C.

Gel preparation
To ease time restrictions, the TA has prepared the agarose gel ahead of time. Remove a
gel carefully and place in the gel tray. Place the tray in the electrophoresis apparatus and
add TBE buffer solution until the gel is completely submersedthere should be a few
millimeters of solution visible above the gel. Take care to position the gel in the
apparatus correctly: the bottom of the gel should be positioned closest to the red
(positive) electrode. Remember: Run to Red! For this experiment 0.8% agarose gels are
used. Think about why a low concentration gel is chosen for better separation in this

Gel loading
1. When the restriction enzyme digests have been carried out for one hour, stop the
reaction by placing the digests on ice. Add 10 L of Purple Loading Dye to each
digest. (This amount corresponds to a 1:5 ratio of loading buffer to sample, since each
enzyme digest was 50L.)
2. Prepare the DNA ladder by diluting 3 L of the stock TrackIt 1 Kb Plus DNA ladder
to 20 L with 1x TBE. It is important to use 1x TBEdilutions with less dense
solutions such as water will float out of the well and to the top of the gel. Do not add
dye to the ladder. Flick the tube to mix thoroughly and spin it down in the
3. With a 20 L micropipet, transfer 15 L of each solution to the wells of the gel. Be
gentle with the tip of your pipet. Insert it carefully into the well, slowly depress the
pipet button to avoid bubbling, and wait for the liquid to be completely dispensed.

Continue depressing the pipet button as you remove the pipet tip from the well, and
from the buffer. Follow the diagram shown below:

1 2 3 4 5 6

Figure 4. The wells of the agarose gel.

1 EcoRI + HindIII digest

2 undigested unknown plasmid
3 DNA marker (TrackIt 1 Kb Plus Ladder)
4 EcoRI digest
5 HindIII digest

After each well is loaded, connect the electrode leads to the power supply of your
apparatus. Make sure that the gel is configured so that the black electrode (which will
become negatively charged) is closest to the wells you just loaded, and the red electrode
(which will become positively charged) is at the opposite end of the gel.
After all of the leads are connected, turn on the power supply; adjust the voltage to 150 V
if necessary. The apparatus may show some condensation it may be easier to track the
progress of the purple dye by looking at it from the side. Stop the electrophoresis when
the purple dye is about 1 cm from the bottom of the gel.

It is important that you read this section carefully before completing the pre-lab.
Disconnect the lid from the chamber and carry the chamber into the side room to the
imaging bench. Remove the gel and place it on the lamp. Before continuing, be sure you
are wearing uv-radiation blocking glasses. This need for uv-protection is unique to this
experiment. Some glasses are provided for this experiment at the imaging desk. Turn off
the light in the room and turn on the transilluminator. The lamp will not turn on unless
the cover is down or the hood is placed over the lamp. Wearing the uv-protecting glasses
or using the orange lens with the black hood, look at the gel and make sure there are 10
bands in the ladder lane. Assuming so, use your smartphone to take a picture of the gel.
Zoom in as far as you can and still see all of the gel.

Cleanup and waste disposal
When you have finished imaging the gel, dispose of it in the Exp. 6 Gel Waste beside the
transilluminator. Pour the used electrophoresis buffer into the Exp. 6 Electrophoresis
Buffer waste in the hood. Your reaction tubes should be disposed of in the Tips Waste at
the original work bench. Turn off ALL equipment, including the voltage supply, water
bath, and transilluminator.

Traditionally, DNA migration lengths have been quantified via rulers in units of cm and
mm. However advances in digital image processing lend themselves readily to agarose
gel analysis. Available online at, ImageJ is a media editing software
package capable of quantifying gel data.
1. Click the ImageJ icon on the desktop to start the program.
2. Once the program is running, drag and drop your image over the ImageJ window to
open it, or use File Open to open your image.
3. Your gel is probably oriented horizontally with the wells on the left side. If not, make
it so by Image and then rotate. This step is not optional- The wells must be on the
left for the intensity profile to make sense.
4. Using the default Rectangular Selections button, draw a rectangle through one of
your lanes. The rectangle should extend from the loading well to well beyond the last
band, be about half the width of the lane, and centered in the lane.
5. Ctrl k will plot the intensity profile of the lane in an x-y coordinate. You should be
able to see individual peaks corresponding to DNA bands. Select the Copy button,
and paste into Excel.
6. Using the down or up arrow to move the selection rectangle, repeat Step 5 for each
lane. Copy them into Excel; delete the superfluous x columns.
7. You should now have an x column corresponding to migration distance in pixels, and
several y columns, each containing the intensity profile for a gel lane.
8. Overlaying the data in a scatter plot will allow for much more accurate determination
of band displacement and consequent DNA fragmentation for your lab report. Please
include this plot in the supplementary information of your lab report.

For your Laboratory Report:
Please answer the following questions in your lab report. This report is to be TYPED,
organized in a logical manner, and due one week after you complete the experiment.
1. Include a picture of your gel image, labeling each lane and showing the position of
each band in each lane.
2. Make a table of the number of fragments in each lane, the distance each migrated, and
the size of each fragment, which you will calculate in Step 5.
3. Find the product information for the TrackIt 1 Kb Plus DNA Ladder you used at
Using this information, identify the DNA fragments in your DNA ladder.
4. Using Eq. 1, construct a plot relating the length of the fragments in the DNA ladder to
the distance they migrated.
5. Using the least-squared fit obtained in Step 4, estimate the size of each of the DNA
samples obtained in the undigested DNA as well as the restriction digests.
6. Construct 3 restriction enzyme maps: for EcoRI, HindIII, and the double digest. Each
map should show each restriction site labelled with the appropriate enzyme, the
number of bases between each recognition site, and the total length of the DNA.
7. In a gel electrophoresis experiment, would an intact plasmid migrate faster, slower, or
the same speed as a plasmid that had been digested by a restriction enzyme at one
position? Why or why not? Apply your answer to your interpretation of your own gel.
8. Determine the resolution you can achieve in this experiment. This is an open ended
question. Defend your answer. What is the smallest distance that you can measure
with the tools you used in this experiment? How many base pairs does this
correspond to? What does this mean about your data?
9. The first DNA sequencing was performed using polyacrylamide gel electrophoresis,
as opposed to agarose gel electrophoresis. What are the differences between these two
types of gels? For what experiments might you choose one over the other? You
should include at least one literature reference in your answer.

Question for Chemical Engineering Students:
Explain Biomanufacturing (what it means, how it is done, advantages, drawbacks,
relation to this experiment, etc). You will need more than a few sentences to get full
credit. Hint: