© 2002 by The International Union of Biochemistry and Molecular Biology
Printed in U.S.A.
BIOCHEMISTRY
AND MOLECULAR BIOLOGY EDUCATION
Vol. 30, No. 5, pp. 306 –308, 2002
Laboratory Exercises
Isolation and Visualization of Nucleic Acid with Homemade Apparatus
PRACTICAL ACTIVITIES FOR SECONDARY SCHOOLS
Received for publication, April 3, 2002, and in revised form, June 18, 2002
Lenira M. N. Sepel and Elgion L. S. Loreto‡
From the Department of Biology, University of Santa Maria, CP 5050,
CEP 97111-970 Santa Maria-Rio Grande do Sul, Brazil
In many developing countries one of the major problems in the teaching science is the lack of adequate
equipment and reagents. One way to solve the problem of the absence of laboratory equipment is to
develop practical classes in which students construct some of the equipment needed for the students to
understand abstract topics in molecular biology. In this article we describe how students can be motivated
to construct a microcentrifuge and electrophoresis apparatus with simple materials. We also give details
of two practical activities (nucleic acid extraction and the separation of DNA and RNA by agarose gel
electrophoresis) that use common non-hazardous household substances and only a few laboratory reagents. Some questions for the students to answer after the practical activities are also presented.
Keywords: DNA isolation, electrophoresis, high school.
The introduction of theoretical concepts unaccompanied by experimental activities is one of the current problems in the teaching of science, because when theory is
divorced from practice learning is only abstract without
direct relevance to the real world. In developing countries
one of the reasons for the emphasis given to the teaching
of theory in secondary schools, and sometimes in undergraduate courses, is the absence of equipment for use in
laboratory activities.
In our experience, challenging students to construct
apparatus is a powerful way to increase their interest in the
structure and isolation of macromolecules such as nucleic
acids and in techniques such as electrophoresis and centrifugation. In this article we describe a homemade microcentrifuge for use in nucleic acid isolation and an electrophoresis apparatus that can be used for the separation of
DNA and RNA, along with a protocol for nucleic acid
extraction that uses readily available non-toxic household
substances. A simple electrophoresis and staining method
is also included.
MATERIALS AND METHODS
Constructing a Microcentrifuge
Materials—
1. PVC pipe cap (50 mm) available from a builders merchant or
plumbing supplier.
2. Electric motor (12 V); we used one from a 12-V hairdryer, but
* This work was developed during the program “PROCIÊNCIAS” for science teaching improvement in secondary school
(supported by Coordenação de aperfeiçoamento de Pessoal de
nivel superior-Fundação de amparo a pesquisa do Rio Grande do
Sul).
‡ To whom correspondence should be addressed. E-mail:
[email protected].
many small motors are available from electrical retailers or
from cheap toys, etc. More powerful 120- or 220-V motors
should be avoided because of safety considerations.
3. Power supply (12 V) and wire.
4. Small plastic jar into which the motor fits tightly.
5. Plastic cap (⫾8 cm) from a 2-liter plastic jar.
6. Microcentrifuge (Eppendorf) tubes.
7. Epoxy resin (e.g. Araldite姞).
8. Sand and cement mixture.
Construction—To make the body of the centrifuge fill the pipe
cap with mortar made from the sand and cement, and insert the
small plastic jar in the mortar, which is allowed to set for 1–2 days.
Then, take out the plastic jar and insert the motor tightly (Fig. 1A).
The rotor is made from the jar cap by cutting four holes (⫾1.1 cm
in diameter) for the Eppendorf tubes and gluing the rotor to the
spindle of the motor (Fig. 1, B and C). The 12-V power supply can
now be attached, and the centrifuge can be tested. Some small
adjustments to the motor may be needed to ensure that the rotor
is balanced properly.
Constructing an Electrophoresis Apparatus
Materials—
1. A plastic box of suitable dimensions (e.g. 10 cm long ⫻ 5 cm
wide ⫻ 5 cm high) to be used as the electrophoretic tank.
2. A smaller plastic box (e.g. 6 ⫻ 4 ⫻ 1.5 cm) to be used to cast
the gel.
3. Stainless steel wire of the type used in dentistry.
4. A 12-V direct current (0.2 amp) power supply, copper wire,
and crocodile clips.
5. Plastic ruler with three-five teeth (0.5 ⫻ 0.5 cm) cut in it.
6. Epoxy resin (e.g. Araldite姞).
7. Agarose gel.
Construction—Two lengths of stainless steel wire are laid in
parallel at the appropriate ends of the box (with one end of each
wire protruding) and glued in place with small drops of epoxy
resin. Care should be taken not to cover the wire, because this
would impede the flow of current. The power supply can be
connected to the free ends of the stainless steel wire with the
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3.
4.
5.
6.
7.
8.
FIG. 1. Homemade microcentrifuge. A, picture showing the
centrifuge parts (electric motor, rotor, and body of the centrifuge).
B, assembled centrifuge ready for use. C, diagram showing the
principal parts of the microcentrifuge.
9.
Drosophila, although other material can be used as a
sample.
Add 0.5 ml of lysis solution to the tube, and homogenize the
sample with a plastic or glass rod. Better homogenization is
obtained if the tip of the rod has the same shape as the
bottom of the Eppendorf tubes.
Incubate the tubes at 60 °C for 15 to 20 min.
Centrifuge the tubes for 5 min in the microcentrifuge and
then carefully transfer the supernatant to a clean tube.
Precipitate the nucleic acids by adding 2 volumes of freezing
ethanol, gently inverting the tubes until the nucleic acids
precipitate and appear as white threads.
Centrifuge the tubes for 1 min to pellet the nucleic acid.
Decant the supernatant, and dry the nucleic acid pellet in air
on the bench for 10 min.
Resuspend the pellet in about 100 ␮l of water (if you do not
have micropipettes, an insulin syringe can be used). The
nucleic acids are now ready to be used in the electrophoresis experiment.
Nucleic Acid Separation by Agarose Gel Electrophoresis
FIG. 2. Apparatus for electrophoresis and nucleic acid banding in agarose gel. A, electrophoresis apparatus (power supply,
electrophoresis chamber, and gel). B, nucleic acid being transferred to the gel well with a pipette. C, gel mold with the combtype well cutter in place. D, agarose gel stained with methylene
blue after 2 h of electrophoresis. Lane 1, nucleic acid from Drosophila flies; lane 2, nucleic acid from onion; lane 3, molecular
weight markers (Invitrogen 1-kb ladder).
copper wire (Fig. 2A). To cast the gel a smaller box (e.g. the top
of a slide box) can be used as a mold, and the wells can be
formed using the ruler with teeth cut in it (Fig. 2C).
Isolation of Nucleic Acid Using Household Materials
The first step in molecular biology experiments usually consists
of the extraction of DNA and/or RNA. Many different protocols are
available for nucleic acid isolation [1, 2], but some of them use
phenol and organic solvents such as chloroform, which are best
avoided in the classroom. Some methods [3, 4] use non-toxic
reagents, but we usually use a method that makes use of household materials, as described in the following protocol for the
isolation of nucleic acids.
1. Make a lysis solution by dissolving 10 ml of an ordinary
kitchen detergent and one soup spoon of salt to 100 ml of
water in a beaker.
2. Put the material from which the nucleic acid is to be extracted into a 1.5 ml Eppendorf tube. Good results can be
obtained with 1 cm2 of onion or three houseflies or about 10
To separate the DNA from the RNA, 0.7% agarose gel can be
used. Use a Bunsen burner or a microwave oven to dissolve the
agarose in TA buffer containing 40 mM Tris-hydroxymethylaminomethane (Tris) in 20 mM acetic acid (to make 100 ml of TA
buffer dissolve 0.5 g of Tris in distilled water, and add 0.15 ml
of acetic acid; no pH adjustment is necessary). Transfer the hot
agarose solution to the gel mold with the toothed-ruler wellformer placed in the position shown in Fig. 2C. It is important
that the teeth of the ruler do not touch the bottom of the gel
mold while the gel is being poured, because if this occurs the
wells will have no agarose at the bottom, and the samples will
leak underneath the gel.
While waiting for the gel to harden, the sample can be prepared for loading. Make a loading solution consisting of a
saturated solution of sucrose in TA buffer containing a drop of
bromphenol blue, 0.5% solution, (normally about 1–2 ml of
loading solution is sufficient), and add 1 volume of loading
solution to 5 volumes of the nucleic acid sample prepared in the
nucleic acids experiment. After the gel has hardened completely (about 20 min), gently take out the well-former and
remove the gel from the gel mold with a spatula, and transfer it
to the electrophoresis tank. Cover the gel with about 2 cm of TA
buffer, and with a pipette or an insulin syringe fill each well with
the loaded nucleic acid sample (Fig. 2B). The density of the
sucrose solution will cause the sample to sink to the bottom of
the well, and the dye will allow it to be seen.
Connect the electrodes to the power supply, with (and this is
very important!) the negative electrode closest to the samples,
and turn on the power supply. After 2 or 3 h (or when the
bromphenol blue has migrated to the middle of the gel) turn the
power supply off, transfer the gel to a staining tray with a
spatula, and cover the gel with a few drops of 0.5% methylene
blue solution. Wait for a half hour and then rinse the gel with
water. At this point the gel will appear a uniform deep blue with
no sign of the nucleic acids. Fill the staining tray with water, and
let the gel soak for about 12–24 h, by which time the methylene
blue should have diffused out of the gel except for the areas
where the nucleic acids are. This is because the dye attaches to
the nucleic acid but not to the agarose molecules. After another
12–24 h the gel will be clear, and the DNA and RNA bands can
be seen very easily (Fig. 2D).
QUESTIONS TO BE ANSWERED BY THE STUDENTS
1. What are the functions of the kitchen detergent and
the salt in the lysis solution?
2. Why is the homogenized sample incubated at 60 °C?
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3. What is the role of the microcentrifuge in the extraction procedure?
4. What occurs when the frozen ethanol is added to the
supernatant?
5. Why was the sample applied to the negative electrode?
6. Why did the RNA migrate faster? What is the importance of the gel in this process?
7. Why does the methylene blue diffusion out of the gel
except from areas where there are nucleic acids?
Acknowledgment—We are grateful to Dr. Robert W. S. P.
Thomas for important suggestions.
BAMBED, Vol. 30, No. 5, pp. 306 –308, 2002
REFERENCES
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Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
[2] R. J. Slader (1986) Experiments in Molecular Biology, Humana Press,
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[3] D. J. Fairbanks, A. Waldrigues, C. F. Ruas, P. M. Ruas, P. J. Maughan,
L. R. Robson, W. R. Andersen, C. R. Riede, C. S. Pauley, L. G.
Caetane, O. M. N. Arantes, M. H. P. Fungaro, M. C. Vidotto, S. E.
Jankevicius (1993) Efficient characterization of biological diversity
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markers, Rev. Brazil. Genet. 16, 11–22.
[4] Genetic Science Learning Center: gslc.genetics.utah.edu/teachers.
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