Plant Biotechnology

LABORATORY EXERCISE
Agarose Gel Electrophoresis

Introduction
Agarose gel electrophoresis is a process that undertakes biochemistry and molecular
biology understandings to identify and analyse DNA and RNA strands. This is done by
separating the genetic material by its size.

The genetic material is placed in the solidified wells of the agarose (a linear polymer
composed of alternating isomers of the sugar galactose) at the cathode end. The
negatively charged nucleic acid molecules move through the agarose matrix with the
assistance of an electric field (electrophoresis). This is because genetic material is
negatively charged, and will move towards the anode when current is passed
through. The shorter molecules migrate faster than the longer molecules.

The use of electrophoresis buffer in the making of the agarose gel is to establish a
constant pH and to provide ions to support the conductivity. If instead water was
used, then the genetic material will not migrate during the electrophoresis.

The amount of voltage used is crucial to the migration of the genetic material. When
increasing voltage is applied to the gel, larger fragments migrate proportionally to
that of the smaller fragments. Thus, the voltage applied is usually 5 volts per
centimetre to the gel. The gel is then immersed in ethidium bromide, a fluorescent
dye that covalently binds (intercalates) between the bases of nucleic acid. Then UV
light is passed through the gel to make the genetic material visible.

Materials
TAE Buffer or TBE Buffer (1x); 6x loading dye (Fermentas/Promega); agarose powder;
standard DNA ladder marker; sterile ddH2O; 10mg/ml EtBr stock solution.

Methodology
1. 0.5g of agarose powder was added with 50ml of 1x TBE in a small Erlenmeyer
flask. This gives 1% gel (The higher the % of gel, the better the separation of DNA
bands).
2. The flask was microwaved until agarose dissolved completely. Solution was
checked occasionally to avoid boiling from occurring.
3. The flask was cooled to 50°C by running it under cold tap water. A comb was
then placed after pouring the agarose.
4. The gel was left to solidify, this process can be fastened by placing the gel rig in a
refrigerator
5. After the gel is solidified, the comb was gently removed by pulling it evenly
upward
6. Gel mould was positioned inside the electrophoresis chamber and covered with
running buffer (1x TBE).
7. DNA samples are mixed with loading dye on a parafilm. (Samples and dyes need
to be diluted accordingly; e.g. 5μl sample + 1μl 6x loading dye or 2μl sample + 3μl
ddH2O + 1μl 6x loading dye).

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8. With the use of a micropipette, the sample mixture and DNA ladder was carefully
loaded into respective wells. Poking the bottom of the gel was avoided.
9. A cover was placed on the electrode box and the gel was electrophoresed at
120V for 45 minutes
10. Once the loading dye reached approximately ¾ of the gel, the power pack was
switched off.
11. Gel was carefully removed from mould and placed it into the staining box
containing EtBr for a few minutes. Gloves were worn because EtBr is mutagenic.
12. DNA stained with EtBr was placed on a transilluminator. The UV light was then
switched on and the DNA bands were visualized.
13. After visualizing of DNA was completed, the gel was disposed in a proper
biohazard waste bucket and the surface of the transilluminator was cleaned with
distilled water.

Results

Done in Biochemistry Laboratory: from right, Lane 2 Muricata paniculata and

Lane 3 Canna glauca. Lane 2 showed a fluorescent clear band and the smear at the
background shows there is DNA denaturation; Lane 3 has vast RNA and protein
contamination.

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Done in Plant Tissue Culture Laboratory: far left, Lane 11 Zinnia elegans showed
clear band of DNA with little denaturation and some protein and
RNA contamination.

Discussions
DNA bands and its quality
The DNA denaturation which causes smear occurs because, whenever a whole plant
genomic is cleaved with an enzyme, usually a smear of DNA fragments can be seen
on agarose gel after electrophoresis. And normally, clear, discrete bands appears on
the background of smear. In many plant genomes, 20-25% of cytosine are
methylated. As a result, when a restriction enzyme is used to cleave the DNA
fragments, various sizes of fragments are formed and the methylated cytosine
produces larger DNA fragments (Hess et al., 1988). Therefore, it is noticeable that
when electrophoresis are being conducted, smaller DNA fragments travels at a faster
rate and larger DNA fragments travel at lower rate.

RNA contamination occurs because RNA has similar structure as the DNA as RNA is
nucleic acid as well. Since RNA is of smaller molecular weight, the RNA fragments
travels at a faster rate compared to DNA. A standard RNase digestion could be done
to eliminate the RNA (Kieleczawa, 2006).

As for protein contamination, we can say that one of the leading factor would be
insufficient phenol extraction. Solvents like phenol or chloroform helps to denature
proteins. Usage of isoamylalcohol helps to separate precipitated proteins and the
nucleic acids remains in the aqueous phase. Kang et al. (1998) reported grinding and
lysis of dry seed and incubation with buffer containing proteinase K gave DNA of
good quality and quantity. Usage of proteinase K helps to overcome shearing of DNA
and loss of yield due to centrifugation by digesting polypeptide into smaller molecule
which can be easily removed by phenol extraction.

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Other ways to improve visualization quality is by increasing concentration of DNA

sample and using SYBR Green I or SYBR Safe instead of ethidium bromide – 25 times
more sensitive and very less mutagenic.

Why no bands?
1. There is a chance where the buffer or agarose used are expired and lose
its activeness.
2. Pellet which supposed to contain the DNA samples are thrown out in previous
experiment.
3. Correct technique during isolation of DNA experiment was not followed.
4. Amount of voltage applied is too low.
5. There is a possibility the percentage of agarose used are too low, thus unable to
produce and visualize DNA samples.

TAE and TBE buffer

Tris-acetate-EDTA (TAE) buffer is a most common buffer solution that consist a
mixture of Tris base, acetic acid, and EDTA. It is used for agarose electrophoresis
analyses of DNA products resulting from PCR amplification, DNA purification
protocols, or DNA cloning experiments, with and without sodium chloride.
Tris-Borate-EDTA (TBE) buffer solution containing a mixture of Tris base, boric acid
and EDTA. It is also often used for agarose electrophoresis analyses of nucleic acids
products.

TAE is best for linear, double stranded, or large pieces of DNA which more than 20kb
but it requires to be replaced frequently for those more than 4 hours gel run times.
TBE is much preferable for separation of smaller DNA fragments such as restriction
enzyme digest. In the other hand, TBE has greater ionic strength and buffering
capacity, allowing us to have sharper resolution compared to TAE, but it is also more
costly and inhibits DNA ligase which causes problems if subsequent DNA purification
and ligation steps are intended as limitation. The use of sodium chloride in TAE may
retards DNA mobility and lead to incorrect interpretations of resulting DNA banding
pattern.

Functions of the chemicals

Agarose (agarose gel) is a polymeric cross-linked polysaccharide extracted from the
seaweed agar. The agarose forms a porous lattice in the buffer solution and the DNA
must slip through the holes in the lattice in order to move toward the positive pole.
This slows the molecule down. Larger molecules will be slowed down more than
smaller molecules, since the smaller molecules can fit through the holes easier. As a
result, a mixture of large and small fragments of DNA that has been run through an
agarose gel will be separated by size.

Agarose was used widely in gel electrophoresis because it gels at a lower

temperature, does not contain the inhibitors of virus growth frequently present in
agar, and has more uniform pore size than that of agar. It also easily poured, does
not denature the samples. The samples can also be recovered. The disadvantages
are that gels can melt during electrophoresis, the buffer can become exhausted, and

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different forms of genetic material may run in unpredictable forms. Increasing the
agarose concentration of a gel reduces the migration speed and enables separation
of smaller DNA molecules. Most agarose gels are made with between 0.7% (good
separation or resolution of large 5-10kb DNA fragments) and 2% (good resolution for
small 0.2-1kb fragments) agarose dissolved in electrophoresis buffer. 1% gels are
common for many applications.

Loading dye plays essential role in obtaining sharp DNA bands. It serves three vital
functions: it is used to terminate enzymatic reactions before electrophoresis (stop
solution - usually provided by EDTA), provide density for loading the sample into the
well (provided by glycerol or sucrose), and it provides the way monitoring the
progress of electrophoresis. EDTA, however it is not sufficient for fully dissociating
DNA-protein complex. Urea, at a concentration of 5M, is the best protein-denaturing
agent because it does not interact with agarose and affecting DNA mobility. Addition
of Ficoll-400 may help increase clarity of bands produced.

Marker is a set of DNA fragments of known molecular sizes that are used as a
standard to determine the sizes of unknown fragments. In addition it can be used to
approximate the mass of a band by comparison to a special mass ladder. The 1kb
ladder with fragment ranging from about 0.5kbp to 10 or 12kbp and the 100bp
ladder with fragments ranging from 100bp to just above 1000bp are the most
frequent. DNA ladders are often produced by a suitable restriction digest of a
plasmid. There are special DNA ladders for supercoiled DNA and RNA.

Ethidium bromide or EtBr is the most common dye used to make DNA or RNA bands
visible for agarose gel electrophoresis. It fluoresces under UV light when intercalated
into DNA (or RNA), of which it decreases DNA mobility up to 15 percent. By running
DNA through an EtBr-treated gel and visualizing it with UV light, any band containing
more than 20ng DNA becomes distinctly visible. EtBr is a known mutagen; however,
safer alternatives are available such as SYBR Green I and SYBR Safe (it is more
expensive, but up to 25 times more sensitive).

Tips
1. Check expiry date of agarose gel and other chemicals before using it to avoid
errors.
2. Avoid creating bubbles when pouring agarose gel into the comb.
3. Pellet to be used must be sterile to avoid contamination and error.
4. Be gentle and caution when inserting the samples into the well and avoid poking
the agarose gel because it may cause leakage.
5. Put on glove as protection before staining process because EtBr is very
carcinogenic.

Conclusion
From this experiment, we become aware and understood on how agarose gel
electrophoresis works.

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References
Barker, K. 2005. At The Bench: A Laboratory Navigator. Cold Spring Harbour
Laboratory Press, New York, USA.

Chawla, H.S. 2002. Introduction to Plant Biotechnology. Science Publisher, Enfield,

New Hampshire, USA.

Hess, W.M., Singh, R.S., Singh, U.S., and Weber, D.J. 1988. Experimental and
Conceptual Plant Pathology. Gordon and Breach, New York, USA.

Henry, R.J. 2008. Plant Genotyping II: SNP Technology. CABI International,
Cambridge, Maasachusetts, USA.

Hames, B.D. and Hinggins, S.J. 1995. Gene Probes: A Practical Approach. Oxford
University Press, New York, USA.

Kang, H.W., Cho, Y.G., Yoon, U.H., and Eun, M.Y. 1998. A Rapid DNA Extraction
Method for RFLP and PCR Analysis From A Single Dry Seed. Plant Molecular
Biology Reporter. 16 (2): 90-94.

Karp, A., Isaac, P.G., and Ingram, D.S. 1998. Molecular Tools for Screening
Biodiversity: Plants and Animals. Kluwer Academic Publisher, Dordrecht,
Netherlands.

Kieleczawa, J. 2006. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones
and Bartlett Publishers, Sudbury, Massachusetts, USA.

Kumar, A and Garg, N. 2005. Genetic Engineering. Nova Science Publisher, New York,
USA.

Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual

Volume 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York, USA.

Surzycki, S. 2003. Human Molecular Biology Laboratory Manual. Blackwell Science,

London, UK.

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
(300909)

http://comp.uark.edu/~mlehmann/Agarose.pdf (300909)

http://www.molecularstation.com/dna/dna-gel-electrophoresis/ (300909)

http://www.plantpath.cornell.edu/glossary/Defs_A.htm (031009)

http://en.wikipedia.org/wiki/TAE_buffer (300909)

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http://en.wikipedia.org/wiki/TBE_buffer (300909)

http://biotech.about.com/od/buffersandmedia/ht/TAE.htm (011009)

http://biotech.about.com/od/buffersandmedia/ht/MakeTBE.htm (011009)