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Printed in U.S.A. Vol. 33, No. 1, pp. 28 –33, 2005
Laboratory Exercises
A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular
biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance
and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves
treatment of various topological forms of DNA with a restriction endonuclease and with a DNA topoisomer-
ase. This session introduces students to the concept of DNA topoisomers, to the properties of different
forms of DNA, and to the activity of restriction endonucleases and topoisomerases toward these forms. The
exercise also involves measuring the size of linear duplex fragments of DNA by comparison of mobility with
a ladder of double stranded DNA of known sizes.
Keywords: DNA topoisomers, restriction endonuclease, DNA gel electrophoresis, DNA topoisomerases, DNA
quantification.
When dealing with DNA there are two fundamental con- the same DNA. This gives the students a feeling for the
cepts that students in molecular biology must have a amount of DNA that can be visualized by this procedure.
feeling for: the small quantities of DNA involved in most The experiment also introduces the concept of topoiso-
manipulations and the topological properties of these mol- mers because, in addition to covalently closed supercoiled
ecules. These concepts are particularly relevant when ex- circular DNA, most plasmid preparations also contain sub-
amining circular and linear DNA molecules of plasmids and stantial amounts of relaxed circles (generated by nicking of
bacteriophage by gel electrophoresis. Students must have one strand) and smaller amounts of linear DNA generated
an appreciation of the relation between the amount of DNA by double-strand breaks. Students are faced with the sit-
and its appearance after agarose gel electrophoresis and uation where a preparation, supposedly containing a sin-
staining with ethidium bromide. They also need to be gle DNA species, shows two or three distinct bands on
aware of the different mobilities exhibited by DNA mole- agarose gel electrophoresis.
cules differing only in their topological form. In addition to In the second part of the practical, plasmid and M13
these practical aspects, the role of DNA topology and its DNA are treated with the restriction endonuclease EcoRI
manipulation in DNA replication, transcription, and other and eukaryotic DNA Topoisomerase I [3], and the reaction
metabolic transactions of DNA is fundamental to an un- products are examined by agarose gel electrophoresis.
derstanding of these topics. Both covalently closed circular (RFI) and single-strand cir-
We have designed a two-part student laboratory exer- cular (SS⫹) M13 DNA samples are treated. The results
cise to illustrate and reinforce these concepts and to pro-
from this part of the practical allow the students to inter-
vide an experimental background to lectures on DNA to-
pret and identify the multiple bands seen in gel electro-
pology and topoisomerases in a third-year undergraduate
phoresis of plasmid DNA in the first part of the experiment.
course in the Institute of Molecular BioSciences. The the-
They can also interpret their results in terms of the ef-
oretical material is based on current biochemistry and
fects of Topoisomerase I and EcoRI on double-stranded
molecular biology texts [1, 2]. About 500 students have
and single-stranded DNA. The reading list for the prac-
used the exercise over the past 10 years.
tical includes material on the functional aspects of DNA
The first part of the experiment involves the quantifica-
tion of plasmid DNA by ultraviolet (UV)1 absorbance fol- topology and its manipulation. This reinforces material
lowed by agarose gel electrophoresis of serial dilutions of on DNA topology contained in the corresponding lecture
course.
The experimental component of the exercise can be
‡ To whom correspondence should be addressed: Institute of completed within two 3-h laboratory periods and is carried
Molecular Biosciences, Massey University, Palmerston North,
New Zealand. E-mail: j.tweedie@massey.ac.nz.
out by groups of two or three students. The total number
1
The abbreviations used are: UV, ultraviolet; TAE, Tris acetate- of students accommodated is only limited by the availabil-
EDTA; TE, Tris-EDTA; DTT, dithiothreitol. ity of equipment for gel electrophoresis.
28 This paper is available on line at http://www.bambed.org
29
OUTLINE OF THE PRACTICAL nt) as described in Sambrook and Russell [4]. The infected cells
First Practical Session are added to 250-ml batches of Luria Bertani medium in 2-liter
flasks and incubated at 37 °C for 5 h with vigorous aeration.
Each group of students is provided with a sample of a Infected cells and phage particles are separated by centrifuga-
plasmid with a single EcoRI site and which is about 3 kbp. tion. Phage particles are precipitated by polyethylene glycol and
We have used pUC118 for the data shown here. They are pelleted by centrifugation. Single-strand circular DNA is isolated
from this pellet by the procedures described in Sambrook and
told the approximate concentration of the plasmid and the
Russell [4]. The covalently closed circular form of M13 DNA is
A260 for double-stranded DNA. After calculating an appro- isolated from the bacterial pellet by the alkaline lysis procedure
priate dilution they prepare a 1-ml aliquot, which is used to also described in Sambrook and Russell [4], and purified by
obtain A260 and A280 readings. They now calculate the equilibrium centrifugation in cesium chloride-ethidium bromide as
actual DNA concentration of their sample and make a described above. Both forms of DNA are suspended at a suitable
series of dilutions that will give known amounts of DNA per concentration (0.5–1 mg/ml) in TE buffer and stored at –20 °C.
10 l of TE buffer from 1 g down to 1 ng. Ten-microliter Enzymes—The restriction endonuclease EcoRI and calf thy-
mus DNA Topoisomerase I are obtained from Invitrogen. DNA
aliquots of the dilutions are mixed with 2 l of loading dye, Topoisomerase I is diluted in topoisomerase storage buffer im-
and the mixed samples were loaded into the wells of an mediately prior to the experiment.
agarose gel. Following electrophoresis, the gels are Agarose Gel Solution—These are made from a 0.7% solution
stained with ethidium bromide, visualized under UV light, of agarose in TAE buffer.
and the image recorded for return to the students for their
interpretation. Sessions 1 and 2
Each group of students is provided with the following:
Second Practical Session • Horizontal gel box with comb (10 well, session 1; 20 well,
In this session, the students are provided with samples session 2). Suitable power supply.
of the plasmid used in the first session and samples of • 100 ml of 0.7% agarose in 1⫻ TAE buffer. (Melted and held
at 55 °C in a water bath. Do not add ethidium bromide to the
double-stranded circular (RFI) and single-stranded circular
agarose.)
(SS⫹) DNA of the bacteriophage M13. • 1⫻ TAE buffer sufficient for gel apparatus used.
Each group of students treats the covalently closed • 100 ml of ethidium bromide staining solution.
circular (ccc, RFI) and the single-stranded circular (SS⫹) • 1.5-ml microcentrifuge tubes.
DNA of bacteriophage M13 with DNA Topoisomerase I • Adjustable pipettors (2–20 l, 20 –200 l, 0.1- 1.0 ml) with
and with the restriction endonuclease EcoRI. Plasmid DNA appropriate tips.
(pUC118) is also treated with EcoRI and with DNA Topoi-
somerase I. Following digestion, the various forms of M13 Session 1
and pUC118 DNA are separated by agarose gel electro-
Each group of students is provided with the following:
phoresis in the absence of ethidium bromide. The gels are
stained following electrophoresis and visualized and re- • 70 l of plasmid DNA (⬃0.5–1 mg/ml) in TE buffer.
corded as for the first session. • 25 ml of TE buffer.
• Spectrophotometer capable of readings at 260 nm.
MATERIALS AND METHODS • 1-ml UV transparent cuvettes.