Restriction Enzyme Digest and Gel Electrophoresis Field Trip
Background Information
Restriction enzymes are proteins that cut double stranded DNA at specific
recognition sites. They were first proposed in the early 1960’s, W. Arber and D.
Dussoix to explain the way bacteriophage (viruses that infect bacteria) could
infect some strains of bacteria but not others. Restriction enzymes are isolated
from bacteria. The bacteria use them as protection against the invasion of
foreign DNA. In 1968, W. Arber and S. Linn and then M. Meselson and R. Yuan
purified similar enzymes that were able to cut DNA, but these early enzymes
cleaved the DNA at random positions. In 1970, H.O. Smith, K. W. Wilcox and T.
J. Kelly purified and characterized the recognition and cleavage site of a more
useful enzyme, Hind II, an enzyme that cut at a specific recognition site every
time. This enzyme was used by Daniel Nathans to cut the circular genome of
Simian Virus 40 (SV40) to generate the first restriction map. The ability to cut
DNA at specific sequences became the first step toward molecular cloning.
In the Restriction Enzyme Digest and Gel Electrophoresis field trip, the
students cut lambda DNA with 4 different restriction enzymes and use gel
electrophoresis to visualize the DNA. Molecular biology laboratory skills and
equipment, as well as laboratory safety, will be discussed and used in this lab.
Lambda DNA is used in this experiment. Lambda is a bacteriophage.
The isolated DNA is linear, 48,502 base pairs long, and has recognition sites for
many different restriction enzymes. It is commonly used for molecular weight,
size markers in gel analysis of DNA, as well a substrate in restriction enzyme
activity assays.
In this lab, we cut lambda DNA with four different restriction enzymes in
the appropriate buffers. This is a restriction digest. Restriction enzymes act
like scissors to cut DNA into pieces. Different restriction enzymes (and there are
hundreds) recognize and cut different DNA sequences. When the DNA is cut,
the sizes of the DNA fragments correspond to the distances (in base pairs)
between restriction sites. The reaction is incubated at the enzyme's optimum
temperature to digest the DNA. This usually takes about 45 minutes. During this
time agarose gels will be prepared.
Purified agarose is a powder that is insoluble in water (or buffer) at room
temperature but dissolves in boiling water (or buffer). As it cools, agarose
undergoes polymerization. The sugar polymers crosslink with each other and
cause the solution to “gel”, much like JELLO. Higher concentrations of agarose
Supported in part by NSF/ATE grants:
DUE #9454555; 9752027
 BTCI, 2000, Madison, WI
give firmer gels. If you were inside a gel, it would resemble a very dense spider
web. If you were a small fragment you could easily crawl through the spaces
between the webs (they are too tough for you to just pull out of the way) but as
you increase in length, it gets harder and harder for you to fit through the spaces.
We usally use a 1.0% agarose gel in sodium borate buffer for this experiment.
The agarose is kept molten at 55C until the gels are poured. Gels are prepared
by pouring the molten agarose into casting trays around a six-well comb and
allowing them to solidify. The comb is removed from the solidified gel and the
DNA is loaded into the wells left by the comb.
The gel box is prepared by pouring buffer into the chambers and placing
the gel in the box. In order to visualize the DNA in the gel, a chemical called
Ethidium Bromide (EtBr) is added to the gel. EtBr intercalates into the DNA and
fluoresces when it is exposed to ultraviolet light. EtBr is a mutagen and must
be handled carefully. We always wear gloves, lab coat and safety glasses when
using EtBr
The digested DNA and some DNA markers will be mixed with a loading
dye containing Tris-HCl, EDTA, 10% Ficoll, and 3 dyes. The samples are loaded
into the agarose gel wells and then electrophoresed by connecting the gel box to
a power supply. Electrophoresis is a laboratory technique used the to separate
charged molecules. DNA is negatively charged and moves, under the force of an
electric current, through the matrix of the agarose. Molecules separate by size,
with the smaller ones moving more rapidly through the gel than the large ones.
After running the gel, the DNA is visualized using medium wavelength
ultraviolet (UV) light (we always wear safety glasses while using a UV light
source). The EtBr binds to the DNA during electrophoresis. When the gel is
exposed to UV light, the DNA bands fluoresce in a distinctive pattern of DNA
fragments, one band for each different size fragment. We take a photograph of
the gel so that it is easier to study the banding pattern.
If you have any questions or would like more information before you bring
your students to the BTCI for this field trip, please give us a call. Alternatively,
bring your questions along and we can discuss them during the lab. We look
forward to seeing you and your group on your scheduled field trip day. Thank
you for your interest in BTCI’s Biotechnology Field Trips program.
Supported in part by NSF/ATE grants:
DUE #9454555; 9752027
 BTCI, 2000, Madison, WI