RESTRICTION ENZYME DIGESTION LAB

Laboratory Overview

1) Set up restriction enzyme digestion reactions

2) Make predictions about results of restriction enzyme digestion reactions
3) Perform gel electrophoresis to analyze results of restriction enzyme digest
reactions
4) Learn about the history of GFP usage in science

Before Lab:
Read Russell, Wolfe, Hertz, and Starr, 2008. Biology: The Dynamic Science. CH 18

Assignment (due at the beginning of next week’s lab)

Formal Lab Report on Restriction Digestion Analysis

Overview: In today’s lab you will use a technique called restriction enzyme mapping to
determine the identity of two different plasmids (small circular pieces of DNA). One of
these plasmids contains the coding sequence for the jellyfish gene product, green
fluorescent protein (GFP), whereas the other plasmid does not. You will write a lab
report detailing your findings from your restriction enzyme digestion reactions.

Next week, you will take the plasmid that contains GFP-encoding DNA and introduce
this plasmid DNA into E. coli bacteria via a process called transformation. In this
manner, you will create transgenic E. coli that express the jellyfish gene product, green
fluorescent protein (GFP), and hence glow green.

Restriction Enzyme Digestion Lab, page 1

BACKGROUND

RESTRICTION ENZYMES
Restriction endonucleases (or restriction enzymes) are bacterial enzymes that act as defense
mechanisms in these organisms. Restriction endonucleases cleave double-stranded DNA
internally, cutting both strands at regions of specific nucleotide sequences that vary from one
enzyme to another. The sequence cut by a restriction endonuclease is its target site (also called
its recognition site). When foreign DNA, such as viral DNA, is introduced into a bacterial cell,
a restriction endonuclease cuts the foreign DNA into shorter pieces, thereby interrupting most of
the foreign genes. This helps defend the cell against invasion by and expression of genes that
could be harmful to the organism. A bacterium protects its own DNA against digestion by its
own restriction enzymes by chemically modifying its DNA soon after DNA replication, usually
by adding methyl groups to bases within the target site of the endonuclease. The enzyme
responsible for protection of the cell’s DNA in this way is a DNA methylase.

In molecular biology, restriction enzymes are used in several ways to modify and manipulate
DNA molecules. One common use is to prepare fragments of DNA from one source to be
combined with fragments of DNA from another source – to construct recombinant DNA.
Another use is to prepare small fragments suitable for nucleotide sequence analysis. Another use
(which is what you will perform in today’s lab) is to provide a rough map of the distribution of
target sites for different restriction endonucleases. This is called constructing a restriction map.

Constructing restriction maps are an important first step in analyzing a cloned DNA fragment or
plasmid. By digesting the DNA with various restriction enzymes, alone and in combination, the
number and relative positions of target sites along the DNA can be determined for each
restriction enzyme. The resulting map can be used to determine information about the DNA
being examined. In this exercise, you will use restriction enzymes to distinguish between two
plasmids to identify the plasmid which contains the GFP coding sequence for use in next week’s
lab.

Plasmids are small circular pieces of DNA that may contain coding sequences for proteins (eg
genes). Some plasmids contain only one gene; others contain multiple genes. Often plasmids
that have genes also contain regulatory sequences upstream of these genes, referred to as
promoters. When these plasmids are introduced into bacteria, the bacteria will express the gene
product downstream of the promoter because the promoter directs transcription of the DNA of
the gene. After transcription, bacteria translate the mRNA into the appropriate protein product.
Next week, you will introduce the pGLO plasmid into bacteria which will cause them to express
the GFP gene product and glow green.

Unfortunately, there was a mix-up during the preparation for this week’s lab. Two different
plasmids, pGLO and pBLU, were placed in separate tubes, but the tubes were not labeled. We
now do not know which tube contains the pGLO plasmid, and which tube contains the pBLU
plasmid. Luckily, the DNA sequence of both of these plasmids is known. In order to determine
which tube contains which plasmid, you will digest each plasmid with the same restriction
enzymes. Because the two plasmids have different DNA sequences, each plasmid will yield

Restriction Enzyme Digestion Lab, page 2

different sized fragments after digestion. You can use the known DNA sequences of the
plasmids to make predictions about the sizes of fragments that will be yielded after digestion
with specific restriction enzymes. You can then compare your results to your predictions to
determine the identity of each plasmid. In order to determine the sizes of your DNA fragments
after restriction enzyme digestion, you will use a technique called gel electrophoresis.

GEL ELECTROPHORESIS
Gel electrophoresis is the most widely used method in molecular biology for separating
macromolecules from one another on the basis of size. It is especially useful for analyzing
mixtures of proteins or of nucleic acids with respect to the presence and relative abundance of
molecules of different sizes, and for estimating the size of purified macromolecules; it can also
be used as a step in purification of a specific protein or a specific length-class of DNA.
Electrophoresis gels for nucleic acids are most commonly prepared with agarose or with
polyacrylamide. In this laboratory, agarose gel electrophoresis will be used to separate DNA
molecules that differ in number of nucleotides and, therefore, in length (size). With a few
changes in details (gel preparation, buffers used, stains applied, etc.), the same overall approach
would used be with a protein solution.

The fundamental principle of gel electrophoresis is that charged macromolecules will migrate
through a gel when an electrical field is applied across that gel. The distance migrated by a DNA
molecule during the time it is subjected to the electrical field is determined by three factors: (1)
the size (length) of the molecule, (2) the electrical field (dimensions and voltage differential),
and (3) the density of the gel matrix. When a sample containing DNA molecules of different
sizes is applied as a mixture to the same position in the gel, all of the molecules are subjected to
the same voltage differential, and all of the molecules are challenged to work their way through
the same gel matrix. This leaves molecular size as the only factor that will determine the final
relative positions of the DNA molecules present in the original mixture.

In environments of pH greater than 7, DNA molecules carry an abundance of negative charges

that are uniformly distributed along the length of each molecule. In this environment, when
DNA is subjected to an electrical field, the DNA molecules will migrate toward the positive pole
of the electrical field. In a gel matrix, the movement of the DNA molecules in response to the
electrical field is impeded by the gel, which acts like a sieve that slows the rate of their
migration. The larger the molecule, the more difficult it is for the molecule to work its way
through the gel; smaller molecules move through the gel faster than larger molecules. As smaller
molecules get ahead of larger molecules, they also get closer to the positive pole, and their
migration accelerates in proportion to their proximity to the positive pole: the closer the
molecule gets to the positive pole, the faster it migrates. Consequently, as smaller molecules of
DNA get ahead of the larger molecules, the relative rate of migration of the smaller molecules is
determined by two factors: (1) their relative ease of working their way through the gel matrix,
and (2) their increasing proximity to the positive pole of the electrical field. This compounding
of influences on the rate of migration results in an exponential relationship between molecular
size and distance migrated.

Restriction Enzyme Digestion Lab, page 3

Gel electrophoresis results in the separation of a mixture of DNA molecules according to
molecular length (size), with the smaller molecules moving ahead of the larger molecules.
Wherever there are many DNA molecules of very nearly the same size, those molecules
accumulate as a migrating “band” that is separated from smaller molecules ahead of them in the
lane of their migration, and larger bands behind them.

The positions of the bands can be visualized by staining the gel with a dye that binds to DNA
molecules, and the size of the molecules in each band can be estimated by comparing the band’s
position (distance migrated) with the positions of DNA molecules of known size. In this
technique, a standard curve serves as the means of estimating the size of the molecules. This is
similar to the use of standard curves for estimating the concentration of smaller molecules, as
you learned in Lab 2 (Spectrophotometry of Proteins) of this laboratory course.

There is, however, one notable difference between the standard curves of Lab 2 and the standard
curve that you will construct in this exercise. In Lab 2, the relationship between Absorbance and
protein concentration (in the useful range of the standard curves) was linear. In this lab, the
relationship between molecular size and distance migrated in an electrophoretic gel is
exponential, as explained above. For this reason, the standard curve that you construct in this
exercise will be plotted on semilogarithmic coordinates so that the curve generated will relate the
logarithm of molecular size to distance migrated plotted on a linear scale.

In order to perform gel electrophoresis, you will mix your RE-digested DNA samples with
“loading buffer”. Loading buffer contains three important components: (1) a dye called SYBR-
green, which binds to DNA and fluoresces when exposed to short-wavelength light (~400 nm),
(2) a dye called bromophenol blue which allows you to view the progression of your sample as it
migrates down the gel, and (3) glycerol, which allows your sample to sink to the bottom of the
well when you are loading the gel.

To determine the size of the products from your RE digests, you will load a solution of DNA of
known sizes (your “markers”) onto your agarose gel along with your restriction enzyme digested
samples. After the samples have been loaded into the gel, the gel will be covered with TBE
buffer at pH 8. Then, the electrophoresis apparatus will be closed, electrical leads will be
attached, and sufficient current will be applied to maintain a voltage differential of
approximately 110 V across the gel. Electrophoresis will continue until the bromophenol blue
indicator dye approaches the positive end of the gel (~ 1 hour). After the gel has been run, your
instructor will disconnect the electrical leads and expose the gel to light of the appropriate
wavelength to cause fluorescence of the SYBR green dye. A picture of this gel can then be
taken, and the location of the fluorescent bands of the DNA markers can be measured. You can
then use these measurements to estimate their length of your RE digestion products (in base
pairs) by comparing the distance migrated by your sample to the distance migrated by the
markers.

Restriction Enzyme Digestion Lab, page 4

PROCEDURE 1A: RESTRICTION ENZYME DIGESTION

1. Sign out a micropipetter from your instructor. Practice pipetting 2.0 μl volumes. This is
somewhat challenging to do accurately. Use the colored water on your lab benches to
accurately measure 2 μl into one tube. BEFORE moving on to step 2, verify with your lab
instructor or TA that the volume is indeed 2 μl. This is critical, because if you do not pipet
accurate volumes, the RE digest will not work!

2. Each pair of students will perform one RE digest assigned by her lab instructor. Label
one 0.5 ml tube, in which you will perform your assigned RE digest reaction.
“MA” for MluI with template A; “HA” for HindIII with template A
“MB” for MluI with template B; “HB” for HindIII with template B

3. Use the table below as a checklist while adding reagents to each reaction tube. Add
components from left to right in the table.

NOTE: The enzymes are in a glycerol solution that is viscous. It is important that you place
only the very TIP of the pipet tip into the enzyme solution. If the tip is placed deep in the
viscous solution, there will be more enzyme on the outside of the tip than within it. Also, too
much glycerol in the digestion mixture can inhibit the activity of the restriction enzyme.

water Buffer DNA template Enzyme

NEB 3 for MluI A or B MluI or HindIII
NEB 2 for HindIII

5 μl 2 μl 8 μl 5 μl

4. Pool and mix the reagents by tapping the tube bottom on the lab bench. Then spin the
tubes quickly (~2 seconds) in the tabletop centrifuge in the lab. Your instructor will
demonstrate use of this centrifuge.

5. Incubate all reaction tubes for 30 minutes at 37oC. During this incubation time, make
predictions about your expected results from the RE digest experiment (Procedure 1B).
These predictions will be necessary for writing your lab report.

6. After 30 minutes, remove your reaction tubes from their temperature controlled area and
proceed to Procedure C: Gel Electrophoresis.

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PROCEDURE B: PREDICTIONS
Generally, when performing RE digestion experiments, the first step is to make predictions about
the expected sizes of DNA fragments after RE digestion. Due to time constraints, we will make
our predictions after setting up our reactions; ideally, you would make your predictions before
you performed an experiment.

Below is a map of each plasmid: pBLU and pGLO. Because plasmids are circular, the number 1
is arbitrarily assigned to the first base of the origin of replication (labeled ori in the diagram
below), and bases are sequentially numbered from that point. Genes (open reading frames) are
diagrammed as blocks and promoter regions are diagrammed as arrows, indicating the direction
of transcription. Even though all plasmids are composed of double-stranded DNA, only a single
line is drawn to represent the double strand of DNA. Restriction enzyme target sites are
diagrammed as lines and labeled with the name of the appropriate enzyme and the base-pair
number at which the enzyme cleaves. The total size of the plasmid (in basepairs) is located in
parentheses in the middle of the plasmid directly underneath the plasmid name. Note that pBLU
is 5,437 basepairs and pGLO is 5,371 basepairs.

pBLU pGLO
1 MluI 1
ori 632 ori

MluI
ampR 1057 MluI
ampR 1135

pBLU pGLO HindIII

(5437 bp) 1465
(5371 bp)
MluI
HindIII ß-gal 1837 MluI
3190 gfp 1666

HindIII
2114

In lab today, you will digest each plasmid separately with either MluI or HindIII. Note that these
enzymes cleave at different locations along each plasmid, and thus will produce different sized
fragments for each plasmid. Based on the sizes of the fragments produced after digestion with
these restriction enzymes, you will be able to determine which tube (A or B) contains the
plasmid pBLU and which tube contains pGLO.

To determine the identity of the plasmids in tubes A and B, you will need to predict the expected
sizes that will be produced after either pBLU or pGLO is digested with either MluI or HindIII.
To do this, start with the pBLU plasmid and the HindIII enzyme. Examine the diagram above.
How many recognition sites are present for the HindIII enzyme on the pBLU plasmid? Based on
this information, how many fragments would you expect to be produced after digestion of the

Restriction Enzyme Digestion Lab, page 6

pBLU plasmid with HindIII? What are the sizes of these fragments? To help you with this task,
you may wish to use the model kits found on your lab benches. Note that in these kits, each
plasmid is represented by a set of tubing, and restriction enzyme recognition sites are represented
by connector pieces. To simulate restriction enzyme digestion, remove the tubing pieces from
each other at the connector sites.

Use the diagrams and the model tubing kits to make your predictions. Record your predictions
in the table below. In the first column of the table, write number of fragments produced by
HindIII digestion, and in the second column, write down the size, in basepairs, of each fragment.

Now examine the pGLO plasmid diagram. How many recognition sites for the HindIII enzyme
are present in pGLO? If HindIII cleaved the pGLO plasmid at all of its recognition sites, how
many fragments would be produced? What are the sizes of these fragments? Use your model
tubing kits and the diagrams to make your predictions, and record these in the table below.

Note the different number and size of the fragments produced by HindIII digestion of pBLU and
pGLO.

Now, repeat this procedure for the MluI enzyme. Predict the number and sizes of the fragments
produced by MluI digestion of the pBLU plasmid. Predict the number and sizes of the fragments
produced by MluI digestion of the pGLO plasmid. Record these predictions in the table below.
You will need these predictions to interpret your data for your lab report.

Predictions for HindIII Digestion:

# fragments Sizes of fragments (bp)
pBLU

pGLO

Predictions for MluI Digestion:

# fragments Sizes of fragments (bp)
pBLU

pGLO

Before leaving lab today, check with your instructor to determine if your predictions are correct.
This is a critical piece of information for you lab report.

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Now that you know the sizes to expect from each digest, how will you visualize the DNA
fragments after they are digested? To determine the size of each fragment, you will perform
agarose gel electrophoresis on the products of the restriction enzyme digestion reactions. To do
this, each restriction enzyme digestion reaction mixture will be mixed with loading buffer and
placed into the wells of an agarose gel. An electric current will be applied, and the negatively-
charged DNA fragments in each well will migrate toward the positive pole of the gel according
to size. Smaller fragments will migrate faster and longer fragments will migrate slower. To
determine the sizes of the fragments from your restriction enzyme digestions, you will need to
compare the distance your unknown fragments migrated to the distance migrated by a known set
of size standards, called a DNA ladder or DNA standard markers.

Let’s examine a sample agarose gel to help us understand how we will determine the sizes of the
DNA fragments from our reactions. On the following page is a diagram of an agarose gel.
There are eight lanes on this gel: lane 1 contains DNA markers (labeled standard), lane 3
contains sample B (labeled B) and lane 5 contains sample K (labeled K). The wells for each lane
of the gel are noted by rectangles, and the DNA fragments are noted by lines. The smallest
fragments have migrated the farthest and are thus located at the end (bottom) of the gel. A key
for the set of standard markers is located on the left side of the gel. You can see that the markers
contained a mixture of DNA fragments that were 20kb, 12kb, 7.5kb, 5kb, 3.5kb, and 1.5 kb. The
1.5 kb fragment, being the smallest, migrated the farthest.

In order to determine the sizes of the fragments in lanes B and K, you must construct a standard
curve using the distances migrated by the standards of known sizes. To do this, measure the
distance traveled (in mm) for each of the standard fragments in the marker lane, and record your
data in the table provided. Then, use the semi-log graph paper available in lab and plot the
distance traveled (in mm) on the X-axis and the size of the DNA fragment (in kb) on the Y-axis
(the log scale). Ask your instructor for assistance if you are not sure how to use semi-log paper.
Draw a line of best fit connecting the points on your curve. Then, measure the distance traveled
by each of the DNA fragments in lanes B and lanes K. Use your standard curve to determine
what size each fragment is. Fill in the data tables on the next page with this information and have
your lab instructor check that you have calculated this correctly. Understanding how to construct
a standard curve using semi-log paper, and use it to calculate DNA fragment sizes is required for
you to write your lab report.

This is an example problem, meant to help you practice constructing standard curves. This
example problem does not go in your lab report.

Restriction Enzyme Digestion Lab, page 8

Example Predictions Assignment: Have your instructor check off that you completed
this assignment before you leave lab today. Do not turn this in with your lab report.

Fragment Distance
size (kb) traveled (mm)
20

12

7.5

5.0

3.5

1.5

Fragment Distance Fragment

traveled (mm) size (kb)

Lane B, #1

Lane B #2

Lane B #3

Lane K #1

Lane K #2

Restriction Enzyme Digestion Lab, page 9

PROCEDURE C: GEL ELECTROPHORESIS
1. Using a micropipetter, add 5 μl loading buffer into your RE digestion tube. Mix the
contents by gently lifting the mixture into the pipet tip and then expelling the mixture
back into the tube – only once.

2. Set your micropipetter to 15 μl. Place a fresh tip on the shaft, and load 15 μl from your
RE digest + loading buffer mixture tube into your assigned lane on a 1.2% agarose gel.
Each group member should write down which sample was loaded into which lane.

3. To determine the sizes of the DNA fragments generated from our RE digestions, we will
compare their distance of migration with the distance of migration of DNA fragments of
known sizes “DNA markers”. The instructor will load 6 μl of DNA markers onto one
lane of each gel.

4. After all wells are loaded, the instructor will close the apparatus, attach the electrical
leads to the power supply, and switch on the current. The power supply is regulated to
deliver sufficient current to sustain a voltage differential of approximately 110 V across
the gel, from the negative pole to the positive pole. Watch for tiny bubbles to rise from
the electrodes, indicating that current is passing through your buffer/gel system. After
about one hour, the indicator dye (bromophenol blue) should be approaching the positive
pole end of the gel, and the current will be switched off and the electrical leads
disconnected.

5. Your instructor will demonstrate for you the removal of a gel from the electrophoresis
apparatus and placement on the visualization light box. Wearing latex gloves, she will
lift the gel tray from the apparatus with the tray slanting downward at one end, where it
will be supported by the instructor’s gloved fingers. This tray, with the gel inside, will be
placed on top of a light source to illuminate the SYBR green dye. Note: When removing
gels from the electrophoresis apparatus, if the tray is not slanted toward the end that is
blocked by the instructor’s fingers, the gel is quite likely to slip out the other end of the
tray onto the bench or the floor of the laboratory. This is invariably disappointing.

PROCEDURE D: PREPARATION OF AGAROSE GEL

1. Because your time in the lab is short, the gel you and your partners use will be ready for
you at the beginning of your lab period. Nevertheless, in order for you to learn the steps
in gel preparation, you and your partners will prepare one gel that will be used by the
next lab section.

2. Fit the gel tray into the casting tray. Make sure that the seal is tight. If you are unsure,
ask your instructor – a tight seal is critical! Insert the well-comb at one end of the tray.

3. Weigh 0.6 g of agarose using the balance, and place this into a 250-ml Erlenmeyer flask.

Restriction Enzyme Digestion Lab, page 10

 4. Measure 50 ml TBE buffer in a graduated cylinder. Pour the buffer into the flask and
mix by gentle swirling.

5. Close the flask with plastic wrap and heat it for approximately 1 min. in the microwave
oven. Watch the mixture as you microwave it to prevent it from “bubbling over” and
spilling. Using a hot-glove to protect your hand, remove the flask and gently swirl the
contents. Examine the contents to be certain that the agarose is completely melted; if not,
return the flask to the oven for additional heating. When the agarose is completely
melted, remove the flask to a water bath at 65oC to cool for a few minutes.

6. When the molten agarose has cooled to 65oC, swirl it gently to be certain that the agarose
is evenly distributed, then pour the contents of the flask into the prepared tray. Rupture
any air bubbles by poking them with a clean pipet tip while the agarose is still molten.

7. Allow the gel to stand until it appears firm and is slightly cloudy, then turn in the gel for
use by the next lab section.

PROCEDURE E: HISTORY OF GFP

In 2008, the Nobel Prize in Chemistry was awarded for, “the discovery and the development of
the green fluorescent protein (GFP)”. Next week in lab, you will start a procedure to create
transgenic bacteria that express the GFP gene product (e.g. the GFP protein), and hence glow
green. The history of the discovery of GFP is rather fascinating, and learning about it will aid in
your understanding of its importance in science. While your gel is running, work in groups of 3-
4 and use the lab computers to learn about five important individuals who made significant
contributions to the discovery and understanding of GFP. Answer the questions on the lab
assignment. Hand in this completed worksheet before you leave lab today.

PROCEDURE F: GEL ELECTROPHORESIS ANALYSIS

1. When electrophoresis is finished, inform your instructor who will help you bring your gel
(in the tray) over to the gel documentation system, and set it up for picture-taking. Your
instructor will take a picture of your gel as it is exposed to short-wavelength light. Each
group member will receive a picture of the gel. Because of the fluorescence of the SYBR
green dye, DNA bands will appear bright white and the gel will appear dark.

2. Take your picture back to your bench and find the bottom of the wells on the gel.
Measure the distance from the bottom of the wells to each bright DNA band. Record
these distances (in mm) in Data Tables provided on the following pages of this handout.

Restriction Enzyme Digestion Lab, page 11

Use the following data tables to collect your raw data during lab. Use this information to help
you in writing your lab report. Do not attach this version of the table to your lab report – to use
this table in your report, reconstruct it for your report. Remember that all tables and figures in
your lab report need titles and captions.

Data Table for Standard DNA (Markers) Agarose Gel

Base pairs/molecule Distance from well to band, in mm

10,000 DO NOT INCLUDE

8,000 DO NOT INCLUDE

6,000 DO NOT INCLUDE

5,000

4,000

3,000 (brighter)

2,500

2,000

1,500

1,000 (brighter)

750

500

250 (may not be visible)

The solution of “Markers” that you loaded into agarose gel contained DNA molecules that varied in size
from 250 to 10,000 bp (base pairs). There should be 13 bands in your Marker lane (although not all may
be visible).

Construct a standard curve for your gel; this will be an important figure in your lab report.
Remember to write a title and caption for this figure. To construct a standard curve:
a. Measure, in mm (estimate to 0.1 mm), the distance migrated by each of the bands in the markers
lane. Measure from the bottom of the well at the beginning of the lane to the leading edge of
each band. Record the distances in the Data Table for your agarose gel.
b. For each band, plot the distance on the X-axis and the molecular size on the Y-axis.
Semilogarithmic graph paper will be provided so that as the molecular size of each band is
plotted, the points will appear on the paper in positions that represent their logarithms; it is not
necessary to look up the logarithm of each molecular size.
c. Draw a line of best fit through all the points that together clearly generate a straight line. Do not
include the points for 10,000, 8,000, or 6,000bp on your graph.

Restriction Enzyme Digestion Lab, page 12

Data Table for Restriction Mapping of Plasmids A and B
Template Enzyme How many Distance (mm) Estimated size of
fragments? each fragment Fragment size (bp)
migrated
A HindIII

B HindIII

A MluI

B MluI

Use your standard curve graph to estimate the size of molecules in any other band on the same gel:

i. Measure the distance the band has migrated (in mm).

ii. Find its distance on the X-axis, follow a vertical line upward until it intersects with the line of
best fit, then follow a horizontal line toward the left to its intersection with the Y-axis. The
value at which this line intersects the Y-axis is the size of the DNA molecules in the band.

iii. Examine the lanes that were loaded with each of the digests, measure the distance (in mm)
migrated by each of the discernible bands, and estimate the sizes of the molecules in each band
(in bp) from your standard curve. Record these values in the data table above. If you wish to
use this table for your lab report, do not attach this one – instead, recreate this table (or a similar
type of table) for your report. Don’t forget to write a title and caption for this table in your
report.

iv. Keep your gel picture and attach it to your lab report, remembering to write a title and caption for
this figure.

Restriction Enzyme Digestion Lab, page 13

 Restriction Enzyme Lab Assignment: The History of GFP

Group Member’s Names: ______________________________________________________

Lab Day/Time/Instructor____________________________

In 2008, the Nobel Prize in Chemistry was awarded for, “the discovery and the development of
the green fluorescent protein (GFP)”. Clearly, this protein has an important place in scientific
history. Go to the website maintained by Marc Zimmer at Connecticut. College:
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm. Read the information on the
home page. Then, click on the “History” link, located on the right side. Five individuals who
were instrumental in the discovery and use of GFP in science are profiled.
1. Next to the name of these individuals below, list a description of their contributions to
GFP (you do not have to write in full sentences). List between 1 and 3 accomplishments
per scientist.
2. Place a star by the individuals who shared the 2008 Nobel Prize.
3. One of the Nobel Prize winners is a faculty member in the Department of Biological
Sciences at Columbia! Circle this individual’s name.
4. Turn in this worksheet before you leave lab today.

Osamu Shimomura

Douglas Prasher

Marty Chalfie

Sergey A. Lukyanov

Roger Tsien

Restriction Enzyme Digestion Lab, page 14

 Restriction Enzyme Lab Assignment:
Lab Report
LAB REPORT GUIDELINES
For this lab, you will write a formal lab report. Although you worked in a group to perform this
lab, each individual should turn in her own assignment. This assignment is due at the beginning
of lab next week.

This lab report should be approximately 5-7 pages, double spaced (including figures, tables, etc).
Remember a longer lab report is not necessarily a better one; good scientific writing is concise.

Include the following in your lab report*:

1. Introduction: Background information to help your reader understand this study. Include the
following:

a. Brief introduction to restriction enzymes (REs) and their use in molecular biology. If you
are writing more than a paragraph, you are probably writing too much.

b. How will you use REs in this study (ie what is the purpose of this study and what are your
hypotheses/predictions)? Note that you will not have a specific hypothesis – instead, you
will be performing an experiment to determine which of two possibilities is true. Since
you did not have any data to guide you before you did this experiment, you should not
make an arbitrary guess about which is true; instead you should make sure that your
experiment will allow you to distinguish between the two possible outcomes.

c. This is a lab report about RE digestion – not GFP – do not include information about the
history of GFP and its usage in science in your lab report (even though you learned about
it during this lab period).

2. Methods: Brief description of the methods you used: Be sure to include information about
volumes and names of reagents used, incubation temperatures and times, etc. You do not need
to include information about how you labeled your tubes, the size of your tubes, etc. This
section will be short (~ 1 paragraph).

3. Results: For this report, your results section will be short. Include the following information.
Don’t forget to include information both in paragraph form and in tables (with
legends/captions).

a. The standard curve you prepared. Don’t forget to include a title and figure legend/caption.

b. The sizes (in bp) of your RE digest fragments (pooled from the class data of all four
digests; calculated using your standard curve). You could put these values in a table.
Remember, tables need titles and captions.

Restriction Enzyme Digestion Lab, page 15

 c. Your gel picture (with a title and caption).

d. A paragraph or two explaining the results from the four different digests (make sure that in
the text you refer to all figures/tables you present).

4. Discussion: Interpret your data. Include the following:

a. Based on your data, which plasmid, A or B, is pGLO and which is pBLU? How confident
are you in your conclusion and why? You may wish to refer to your standard curve in this
discussion; however, there is likely no need to write more than one or two sentences about
your standard curve in your discussion.

b. Discuss the significance of this study (e.g. why is it important to know which plasmid is
pGLO and which is pBLU? Hint: The answer to this question includes what you plan to
do with the pGLO plasmid in the coming weeks).

c. Did you find anything surprising or unexpected (such as fragment sizes that were not
predicted)? If so, why do you think you found these unusual results?

5. References. Cite all outside references if you used any. You should cite the lab manual and
at least one additional outside reference (like a text book, reliable website, etc). Make sure
that if you list a reference in this section, you have an in-text citation somewhere in your lab
report. Never include direct quotations from your references; always paraphrase in your own
words. Never cite Wikipedia – it is not a reliable academic source.

6. Don’t forget to include an informative title and your name/lab section with your report.

*NOTE: The above instructions are meant as guidelines to help you prepare your report. They are,
by no means, a comprehensive list of everything that should go into the report.

Restriction Enzyme Digestion Lab, page 16