Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
Experiment 6
RESTRICTION ENDONUCLEASE DIGESTION AND DNA GEL ELECTROPHORESIS
Objectives:
1. To learn the basics about Restriction Endonucleases and their action.
2. To perform a virtual restriction digest and map Lambda genome using an Internet based
Bioinformatics based software.
3. Learn and perform 'wet' restriction digestions of Lambda genome.
4. Learn and perform agarose gel electrophoresis to separate the DNA fragments produced by
restriction digests.
5. Learn how restriction endonuclease produced DNA fragments can be used for mapping the
DNA of an organism (Lambda genome).
Introduction/Background
What are restriction endonucleases (REs)? :
Restriction enzymes recognize a specific nucleotide sequence and produce a break across both
strands of the DNA. Depending upon the recognition sequence and exact position of their action,
the result could be a straight or staggered cut. In a lot of cases the nucleotide sequence in the
recognition site is palindromic.
Examples of palindromic words: mom, dad, live not on evil
The palindromic sequence reads the same in the 5' to 3' direction on both strands of DNA. e.g.
Eco RI (isolated from E. coli ) : GAATTC
This particular enzyme and a lot of other enzymes produce staggered cuts resulting in single
stranded complementary tails called sticky ends. Other examples of enzymes that produce sticky
ends are: HindIII, BamHI, XhoI.
While most restriction enzymes recognize hexanucleotides or tetranucleotides, several others
recognize different lengths of nucleotides. If a restriction endonuclease recognizes a
hexanucleotide sequence, then it would be expected to cut genomic DNA at random
approximately every 4100 bp (46). For tetranucleotide this number would be 44= 256 bp.
What are the uses of REs ? :
Restriction endonuclease digestion is one of the important methods of molecular biology.
Restriction enzymes can be used for various purposes including, but not limited to: mapping,
diagnostics, DNA cloning. Generally, the first step in exploring a cloned DNA fragment is to
construct its restriction map. By digesting the DNA with various restriction enzymes, alone and
in combination, we can determine the number and relative positions of cutting sites along the
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
DNA for each restriction enzyme. The restriction map can be used to determine the smallest
restriction fragment containing an intact gene or to provide restriction fragments suitable for
subcloning .
The DNA fragments from two different sources but cut with the same enzyme (which produces
sticky ends) can be annealed to form recombinant molecules. T4 DNA ligase is used to join these
fragments.
REs can also be used for identification of DNA fragments, plasmids and genomes.
Storage and Handling:
Restriction enzymes, like many enzymes, are most stable at cold temperatures, and lose activity
if they are warmed for any length of time. Maintaining these enzymes in good condition is
critical to the success of the experiments.
 Storage at -10o C to -20o C in NON-frost free freezer
 Use of coolers/ice on benches
 Use of gloves
 Storage in 50% glycerol
Optimal reaction requirements:
 Enzyme concentrations
 One unit = the amount of enzyme necessary to digest 1 g of Lambda DNA in 1 hour at
370C.
 Buffer requirements. 10 x stocks.
 Temperature requirements
 glycerol concentrations <5% ?
 Incubation time
DNA handling
Restriction map of lambda genome
Setting up a restriction digest
Incubation
Agarose gel electrophoresis:
Based on the principle of separating molecules by their attraction to an electrical charge and
differences in their molecular sizes. Because DNA contains a negative charge resulting from the
phosphate groups linking the deoxyribose backbone, it will migrate toward the positive pole
when placed in an electrical field. Agarose is a highly purified form of agar and when solidified
will make a network through which DNA must move. The result is that large DNA fragments
will migrate more slowly through, the agarose, whereas smaller DNA fragments will migrate
more rapidly. These different rates allow for the separation of a mixture of DNA fragments by
their molecular weights (sizes !). The molecular weight of a particular DNA can be used to
determine the approximate number of nucleotides in the DNA. Other factors such as linear
versus circular DNA, supercoiling, and ionic strength of the buffer can also influence the
migration of DNA.
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
Materials/ Reagents/ Supplies
Per Table
 Ice in the bucket
 Test tube rack with 10 eppendorf tubes (autoclaved).
 100 l of unmethylated Lambda DNA solution (125 g/ml ). On ice.
 Restriction endonucleases: Bam HI (5 l), Eco RI (5 l), and HindIII (5 l). On ice or freezer
 Appropriate 10x stock endonuclease digestion buffers. On ice. 10 l per each enzyme.
Bam HI: REact 3 or High Salt;
Eco RI: React 3 or High Salt;
Hind III: React 2 or MediumSalt (buffer B);
Eco RI/Bam HI: React 3 or High Salt
 1000 l autoclaved distilled water in an eppendorf tube. On ice.
 20 l of 10x DNA loading Dye buffer (See appendix)
 25 l of Molecular Weight Marker (1 kb ladder) solution made in 1x DNA loading Dye
Buffer. 0.2 g/l concentration. On ice
 Agarose "baby" gel apparatus with combs and glass/plexiglass plates/trays.
 Power supply
 150 ml flask (not required if pre-melted 0.8% agarose is provided)
 Stirring bar (not required if pre-melted 0.8% agarose is provided)
 Weighing boat (not required if pre-melted 0.8% agarose is provided)
 Spatula
 1 liter electrophoresis buffer (1x TBE, made from 10x stock, see appendix)
 50 ml graduated cylinder
 Magnetic stirring hotplate (not required if pre-melted 0.8% agarose is provided)
 Insulated "hot" glove
 Pipetteman 1-5 l range. Pipetteman 5-25 l range.
 Supply of appropriate pipette tips (autoclaved)
 1 plastic or glass tray for staining the gel
 Plastic or latex gloves
 Safety Glasses
 Permanent Marker.
For the Class
 Analytical balance (not required if pre-melted 0.8% agarose is provided)
 Eppendorf microfuge
 Agarose powder (not required if pre-melted 0.8% agarose is provided)
 Autoclave or masking tape
 UV transilluminator and appropriate camera with film.
 Vortexer
 Waste container for Ethidium Bromide
 Ethidium Bromide Stock (10 mg/ml)
 Water bath set at 37 degrees Centigrade with a float for eppendorf tubes
 Water bath set at 65 degrees Centigrade with a float for eppendorf tubes
 Saran Wrap
 Supply of distilled water for ethidium bromide staining and destaining.
 Computer, projector, Internet connection.
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
Instructions
Lambda is bacteriophage that has genome approximately 50 kilobases (48.5 kb)) in length
and that consists of linear (!!), double-stranded DNA. Because of its size and the fact that it
can be obtained commercially in a nonmethylated form, Lambda DNA is ideal for
demonstrating restriction analysis of DNA. Today you will analyze restriction fragments of
Lambda DNA cut by two different restriction endonucleases, Eco RI and Hind III.
IMPORTANT: Unless specifically instructed, perform all the procedure at 0 - 40C (on ice).
A. Restriction Endonuclease digestion of Lambda DNA
1. Your instructor will assign each person to use one of the three restriction enzymes. Different
members of your group should be assigned different enzymes so that everyone will see the
different fragment patterns after gel electrophoresis. In addition, your group will also perform
a double-digest using both Eco RI and Bam HI enzymes in one reaction.
2. Use a micro-Pipetteman to transfer 8 l of the Lambda DNA solution (1g total to a 1.5-ml
Eppendorf tube.
3. Next, place into the Eppendorf tube 2 l of the 10x digestion buffer for your restriction
enzyme and mix on a vortex mixer. Make sure to use the buffer with appropriate salt
concentration recommended for the enzyme being used.
Bam HI: REact 3 or High Salt;
Eco RI: React 3 or High Salt;
Hind III: React 2 or MediumSalt (buffer B);
Eco RI/Bam HI: React 3 or High Salt
4. Pipette pre-calculated volume of autoclaved D.W. into the tube
5. Transfer 1 l (1-10 units) of one of the restriction enzymes to the Eppendorf tube. For double
digests pipette 1 l each of the enzymes. Use a fresh pipette tip every time. After addition of
the enzyme(s), mix the contents (gentle tapping only!) and incubate at 370C for 1 hour.
Single Digests:
Lambda DNA ( 125g/ ml):
10x Buffer:
Autoclaved D.W.
Restriction Enzyme(1-10 units/l):
Total
Double Digests:
8 l
2 l
9 l
1 l
20 l
Lambda DNA ( 125g/ ml):
10x Buffer:
Autoclaved D.W.
Restriction Enzyme-I (1-10 units/l):
Restriction Enzyme-II (1-10 units/l):
Total
8 l
2 l
8 l
1 l
1 l
20 l
While the DNA is being digested, start preparing your agarose gel following the procedure
outlined below in section B.
6. At the end of 1 hour (o/n digestion is okay), remove your Eppendorf tube from the 370C
incubator or water bath and add 2 l of 10x loading dye buffer, and then mix. If some of the
mixture remains on the side of the tube, gently tap the tube on the table or spin the tube in the
eppendorf microcentrifuge for 30 seconds to force the solution down to the bottom of the
tube.
7. Proceed with the gel electrophoresis procedure given below.
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
B. Preparing the gel
1. Weigh 0.4 g agarose in the weighing boat and carefully add the agarose to the 150-ml flask,
making sure that none of the agarose touches the sides of the flask.
2. Measure 50 ml electrophoresis buffer (1x TBE) in a 50-ml graduated cylinder and then
carefully pour the buffer into the 150-ml flask containing the agarose. This will make a 0.8%
agarose gel. Next, add the magnetic stirring bar to the flask and place the flask on the
magnetic hotplate. Turn the hot- plate to high and the stirrer to a position such that the
solution is being completely mixed. Heat the mixture until it starts to boil. The mixture will
appear a cloudy white at first, but it will be clear when ready to use.
Alternatively, your instructor will demonstrate how to heat the agarose solution in a microwave.
3. While the agarose is heating, rinse the electrophoresis apparatus with distilled water if it
appears dirty, and then dry it with paper towels. Be careful not to damage the thin platinum
wire electrodes that are at the bottom of each end of the apparatus.
4. Observing the inside of the electrophoresis apparatus, you will see a central platform, which
is open on each end. This area will hold the gel when it is poured. Place the glass plate
provided on the bottom of the platform, and, using the tape that is provided, tape the two
open ends of the gel platform. Rub your fingernail across the tape where it meets the
platform to ensure a tight seal. (Some electrophoresis chambers have the platform at the
bottom of the apparatus and have the ends sealed with plastic or metal inserts. If this is the
case, your instructor will demonstrate how to set this up.)
5. After taping the ends of the platform, place the Teflon spacer comb on the platform using the
holes provided. Place the comb such that the spacers are as far away from the edge of the
platform as possible. In addition, make sure the comb teeth do not touch the bottom of the gel
apparatus. After the agarose is poured and the gel is solidified, the comb will be removed.
The spacers, will create wells to which DNA samples can be added. Most combs have eight
spacers, so that eight wells will be present in the gel.
8. Make sure that the electrophoresis apparatus is resting level. When the agarose just starts to
boil, remove it from the hotplate (or microwave) using the insulated glove and carefully pour
it into the center of the gel platform. Some gel formers require that the agarose be cooled to
avoid warping. If this is the case, your instructor will inform you. Pour enough solution so
that the agarose is even with the plastic side boundaries of the platform. Make sure there are
no strong air drafts that would cause ripples in the gel, and then allow the gel to solidify. The
gel will become slightly opaque when it has solidified. This will require approximately 15
minutes.
7. Once the gel is solidified, remove the tape from the edges of the platform and fill the
chamber with electrophoresis buffer so that it completely covers the gel by approximately
one cm. Allow the gel to sit in the buffer for a minute or two, and then remove the comb by
pulling up evenly on both ends of the comb. The buffer should fill the wells when the comb
is removed. If a bubble is present in a well, use the Pipetteman to blow buffer into the well
forcing the bubble out. The gels may sit several hours in this state. Note: If the comb is
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
removed before the gel is sufficiently solidified, the wells may be damaged, resulting in
sample leakage from the wells.
C. Gel Loading and Electrophoresis
1. Your DNA sample has been combined with approximately 2l of the 10x loading dye buffer
for a total volume of 22 l and is in your 1.5-ml Eppendorf centrifuge tube. Incubate the
tubes containing your samples at 650C for 5 minutes, spin them for 15 seconds without
any delay and leave them at room temperature. (Your instructor will explain the detailed
reason for this step which involves disruption of COS Hydrogen bonding).
2.
Adjust your Pipetteman to 22 l and attach a clean tip. (Your instructor will demonstrate the
proper use of the Pipetteman,) Depress the plunger on the Pipetteman to the first stop; then,
place the tip to the bottom of the DNA sample and slowly release the plunger. This action
will allow 22 l of the DNA sample to be drawn into the pipette tip. Starting from one side of
the gel and proceeding toward the other for each member of your group, position the tip of
the pipette at the top of a well and slowly depress the applicator button to the first stop
position. Because the gel dyes contain compounds denser than the buffer, the DNA sample
will flow to the bottom of the well. Do not depress the applicator button to the blow-out
position; doing so would force air into the well, causing the sample to be ejected.
3. When all members of your group have placed their samples into the wells, one of the
members can load 10 l of the molecular weight marker into the last well. Put the chamber
lid onto the electrophoresis chamber. Attach the black wire to the negative pole and the red
wire to the positive pole of the chamber, and then attach the other ends to the respective poles
of the power supply. Note: DNA is negatively charged; therefore, the positive pole should be
at the far end from the sample wells.
4. Set the power supply at 75 V and turn it on. Warning: You are dealing with an electrical
source. Do not touch the buffer or unit while the power is on. As the sample migrates through
the gel, you will begin to see a blue dye band and a blue-green dye band.
Let the gel electrophoresis proceed until the first band is two-thirds of the way through
the gel and the second is one-third of the way through. This should require approximately 2
hours. Increasing the voltage will decrease the amount of time required for migration through
the gel, but it will also decrease the resolution of the DNA bands.
D. Staining and Photography of the Gel
1. Place approximately 200 ml distilled water into a container and add 30 l ethiduim bromide
stock solution (10 mg/ml). Gently swirl the container to mix the stain. Warning: Always
wear gloves when working with ethidium bromide because it is a potent mutagen and
potential carcinogen !!
2. Because it is desirable to flip the gel over for viewing and photographing, it becomes easy to
confuse which is the first lane and which is the last lane of DNA. To avoid this potential
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Instructor:
Paramjeet S. Bagga
3.
4.
5.
6.
7.
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Lab Protocols
Cell & Molecular Biology
SBIO 406
problem, asymmetry can be introduced into the gel by making a small notch at one side of
the gel bottom with a spatula.
Carefully lift the gel from the chamber platform using the glass plate to support the gel.
Transfer the gel to the ethidium bromide solution by allowing the gel to slide off the glass
plate gently into the solution. Note: Agarose gels are flimsy and can easily tear, so handle
them with care.
Place the container on a shaker at low speed for 20 minutes.
After the 20-minute staining period, pour the ethidium bromide solution into the waste
container provided, being careful not to let the gel slide out of the container.
Gently pour approximately 200 ml distilled water over your gel and wash the gel for 10
minutes at low speed on the shaker. After the 10-minute wash, discard the distilled water into
the waste container.
Using a spatula, carefully lift one end of the gel and slide your fingers under it. Lift the gel
out of the container and flip it over; then, place it on a piece of Saran Wrap. Since the DNA
was at the bottom of the well, flipping the gel over places the DNA closest to the camera for
observing and photographing.
While it is still on the Saran Wrap, place the gel on the ultraviolet transilluminator, which has
been positioned behind a plexiglass shield to reduce your exposure to UV light. Put on plastic
safety glasses to protect your eyes further from the UV light and then turn on the
transilluminator. Ethidium bromide will fluoresce orange anywhere it has intercalated with
DNA. Your instructor will demonstrate how to use the camera and photographic apparatus, if
you are photographing the gel. If not photographing the gel, then quickly draw the banding
pattern(s) in your notebook.
E. Analysis and Documentation of the Results:
1. Paste the gel picture in your lab note book, label all the lanes and known molecular sizes.
2. Measure the migration distance of all the major bands as instructed and plot a graph between
Molecular weight vs. Migration distance for the Molecular weight marker lane.
3. Determine the Molecular sizes of all the Restriction DNA fragments separated by
electrophoresis (for all, Bam HI, Eco RI, HindIII and double digests separately).
4. Compare your results with the virtual digests explained below in the next section.
5. Explain any discrepancies or unresolved bands
6. Explain what did you learn from this exercise.
F. Virtual Restriction digestion of Lambda DNA
Your instructor will demonstrate the use of an Internet based Bioinformatics program called
Webcutter (http://www.firstmarket.com/cutter/cut2.html), that can perform virtual restriction
digests of any given DNA sequence within seconds and provide you with a very valuable
information.
Submit the lambda genome DNA sequence for restriction analysis for Bam HI, Eco RI and
HindIII enzymes that you used for 'wet' lab analysis. Print out the results and paste them in
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
your lab notebook. Describe and explain the results. Compare the expected sizes of
Restriction DNA fragments thus obtained with your actual 'wet' lab results obtained above
Answer the following Questions
Why do DNA bands migrate at different rates ?
Did any of the bands appear the same in the two restriction-cut DNAs ?
If so, what does this similarity suggest you ?
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