Gel Electrophoresis

Gel electrophoresis is the process by which scientists can sort pieces of DNA from
PCR product or cut DNA with restriction enzymes by size. An agarose or polyacrylamide gel
is loaded with the DNA fragments and current is passed through the gel. Since DNA is
negatively charged, it will migrate towards the positive pole. The DNA will not migrate at the
same rate, however. Larger pieces of DNA collide with the gel matrix more often and are
slowed down, while smaller pieces of DNA move through more quickly. Since different genes
have different nucleotide sequences, restriction enzymes will cut them at different places,
generating different size DNA fragments. By using gel electrorphoresis, biologists can tell
which gene is which based upon the sizes of the fragments generated when a gene is
treated with a restriction enzyme.
Electrophoresis is a technique used to separate and sometimes purify
macromolecules - especially proteins and nucleic acids - that differ in size, charge or
conformation. As such, it is one of the most widely used techniques in biochemistry and
molecular biology. When charged molecules are placed in an electric field, they migrate
toward either the positive (anode) or negative (cathode) pole according to their charge. In
contrast to proteins, which can have either a net positive or net negative charge, nucleic
acids have a consistent negative charge imparted by their phosphate backbone, and migrate

Figure 1. Electrophoretic migration of charged particles.

toward the anode (Fig. 1).

Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most
commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The
gel is immersed within an electrophoresis buffer that provides ions to carry a current and
some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which have
attributes suitable to particular tasks:
Agarose is a polysaccharide extracted from seaweed (Fig. 2). It is typically used at
concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel.

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 Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer
solution, melt it by heating, and pour the gel. It is also non-toxic. Agarose gels have a
large range of separation, but relatively low resolving power. By varying the
concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be
separated using standard electrophoretic techniques.

Figure 2. Agarose structure unit

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer

chains is ictated by the concentration of acrylamide used, which is typically between 3.5
and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose
gels. Because oxygen inhibits the polymerization process, they must be poured between
glass plates (or cylinders).Acrylamide is a potent neurotoxin and should be
handled with care! Wear disposable gloves when handling solutions of acrylamide, and
a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but
polyacrylamide gels should also be handled with gloves due to the possible presence of
free acrylamide. Polyacrylamide gels have a rather small range of separation, but
very high resolving power. In the case of DNA, polyacrylamide is used for separating
fragments of less than about 500 bp. However, under appropriate conditions, fragments
of DNA differing is length by a single base pair are easily resolved. In contrast to
agarose, polyacrylamide gels are used extensively for separating and characterizing
mixtures of proteins.

Table 1. Concentrations of Agarose and Acrylamide Giving Optimum Resolution of DNA

Fragment.
Agarose Polyacrylamide
% Resolution (kb) % Resolution (bp)
0.9 0.5 - 0.7 3.5 1000 - 2000
1.2 0.4 - 6.0 5.0 80 - 500
1.5 0.2 - 3.0 8.0 60 - 400
2.0 0.1 - 2.0 12.0 440 - 200

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Preparation and Examination of Agarose Gels

Preparation of an agarose gel

1. Clean and dry the glass plate then assemble the tray on the gel molder with the
appropriate gel comb. Set the mold on a horizontal section of the bench.
2. Prepare sufficient electrophoresis buffer (usually 1x TAE or 0.5x TBE) to fill the
electrophoresis tank and to prepare the gel. Add the correct amount of powdered
agarose to a measured quantity of electrophoresis buffer in an Erlenmeyer flask. The
buffer should not occupy more than 50% of the volume of the flask.
3. Loosely cover the neck of the flask. Heat the slurry in a microwave oven until the
agarose dissolves. Heat the slurry for the minimum time required and gently swirl the
bottle from time to time to make sure that any grains sticking to the walls enter the
solution.
CAUTION: Take care-the agarose solution can become superheated and may boil
violently if it has been heated for too long in the microwave oven.
4. Cool the solution to 60OC, and, if desired, add ethidium bromide (from a stock solution
of 10mg/ml in water_ to a final concentration of 0.5 ug/ml and mix thoroughly.
CAUTION: Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves
should be worn when working with solutions that contain this dye. After use, these
solutions should be decontaminated.
5. Pour the warm agarose solution into the mold. The gel should be between 3mm and
5mm thick. Check to see that there are no air bubbles under or between the teeth of the
comb (Fig. 3).
6. After the gel is completely set (30-45 minutes at room temperature), carefully remove
the comb and mount the gel in the electrophoresis tank.
7. Add just enough electrophoresis buffer to cover the gel to a depth of about 1mm.
8. Mix the samples of DNA with the desired gel-loading dye. Slowly load the mixture into
the slots of the submerged gel using a disposable tip attached to an automatic
micropipettor.
9. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate
toward the anode. If the leads have been attached correctly, bubbles should be
generated at the anode and cathode and, within a few minutes, the bromphenol blue
should migrate from the wells into the body of the gel.
10. After the electrophoresis, turn off the electric current and remove the leads and lid from
the gel tank. If ethidium bromide was present in the gel, examine the gel by ultraviolet
light and photograph the gel.

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 prepare the gel-casting tray place the comb materials and chemical for
agarose gel preparation

melt agarose in microwave add ethidium bromide

Pour gel after cooling allow the gel to harden for at least 30 min before use

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dot dye and DNA on parafilm load onto the gel place in the tank

Set the power supply to desired voltage then run

Take a picture of the gel for documentation sample of PCR product on agarose gel

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Additional information to agarose gel electrophoresis
The molecular size of the DNA. Molecules of linear, duplex DNA, which are believed to
migrate in an end-on position (Fisher and Dingman; Aaij and Borst) travel through gel
matrices at rates that are inversely proportional to the logarithm of their molecular weights
(Helling et al.).
The agarose concentration. A DNA fragment of given size migrates at different rates
through gels containing different concentrations of agarose. There is a linear relationship
between the logarithm of the electrophoretic mobility of DNA () and gel concentration (),
which is described by the equation:

log = log O - Kr
where O is the free electrophoretic mobility and Kr is the retardation coefficient, a constant
that is related to the properties of the gel and the size of the migrating molecules. Thus, by
using gels of different concentrations, it is possible to resolve a wide-range of DNA
fragments.
The conformation of the DNA. Closed circular, nicked circular and linear DNA of the
same molecular weight migrate through agarose gels at different rates. The relative
mobilities of the three forms are dependent primarily on the agarose concentration in the gel
but are also influenced by the strength of the applied current, the ionic strength of the
buffer, and the density of superhelical twists in the DNA.
The applied current. At low voltages, the rate of migration of linear DNA fragments is
proportional to the voltage applied. However, as the electric field strength is raised, the
mobility of high-molecular-weight fragments of DNA is increased differentially. Thus, the
effective range of separation of agarose gels decreases as the voltage is increased. Gels
should be run at no more than 5 V/cm.

Polyacylamide gel electrophoresis

Polyacrylamide Gel Electrophoresis (PAGE) is one of the most important analytical
methods in molecular biology. Highly porous gels can be prepared through the
polymerization of acrylamide and a cross-linking agent such as bis-acrylamide. This free
radical polymerization uses ammonium persulphate as the inhibitor and TEMED (N,N,N',N'-
Tetramethylethylenediamine) as the catalyst. Caledon acrylamide and bis-acrylamide are
extremely pure reagents that have been prepared by recrystallization of already pure
material. Ratio 19:1 is ideal for the separation of nucleic acids. Ratios 29:1 is for DNA and
proteins, and the 37.5:1 ratio (30:0:8) is for protein electrophoresis.

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 The advantages of purification on denaturing polyacrylamide gels are speed,
simplicity, and high resolution. Denaturing polyacrylamide gels can resolve oligonucleotides
from 2 to 300 bases, depending on the percentage of polyacrylamide used (see Table). This
method is thus useful not only for isolating chemically synthesized deoxyribonucleotides but
also small RNAs or other single-stranded oligonucleotides. After gel setup, samples are
loaded onto a urea-based denaturing gel, separated by electrophoresis, and finally
recovered from the crushed gel slice.

Table 2. Concentrations of Acrylamide Giving Optimum Resolution of DNA Fragments Using

Denaturing PAGE
Acrylamide Fragment sizes migration of migration of
(%) separated (bases) bromophenol blue xylene cyanol
(bases) (bases)
30 2 to 8 6 20
20 8 to 25 8 28
10 25 to 35 12 55
8 35 to 45 19 75
6 45 to 70 26 105
5 70 to 300 35 130
4 100 to 500 ~50 ~230
Source: Maniatis et al., 1975.

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Forming of polyacrylamide gel

Polyacrilamide is a polymer made of acrylamide (C3H5NO) (Fig. 4) and bis-acrilamide (N,N-methylene-bis-acrylamide C7H10N2O2). Bis-Acrylamide

polymerizes along with acrylamide forming cross-links between acrylamide chains. This is a
synthesize gel that have uniform pore size, no reaction with other chemicals, viscosity,
stable in very length of pH and temperature and have wild ionic strength. We can design the
pore size in polyacrylamide gel by adjusted the concentration of acrylamide and bis-
acrylamide. TMED (N,N,N,N-tetramethylethylenediamine) is catalyst in the polymerization
forming of acrylamide and bis-acrylamid (Fig. 5).

Figure 4. Structure of acrylamide and Bis-acrylamide

Figure 5. Polymerized acrylamide gel

Polyacrylamide gel electrophoresis protocol

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Polyacrymide gel preparation
1. Wipe a chamber once with 95% EtOH.
2. Wipe a chamber with clean view solution and let it dry.
3. Wipe a gel plate with 95% EtOH for 3 times.
4. Wipe a gel plate with 700 ul of bind silane solution, let it dry then clean with 95% EtOH.
Bind silane solution: 3 ul bind silane + 1 ml of 0.5% acetic acid in 95% EtOH.
5. Set the gel chamber and gel plate carefully.
6. Prepare acrylamide gel.
50 ml of 4.5 acrylamide gel + 70 ul TEMED + 350 ul of 10% APS, gentle shake and use
immediately.
7. Poor gel into the gel-set carefully then make the well by push the comb into the top of
the filled gel-set.
8. Let gel setting for 30 minute to 1 hour.
(Refer to Figures below)

Gel running
1. Pre-run by add 1X TBE buffer (1.5 l for 1 gel-set) then remove comb and clean up the
well.
2. Pre-running is done by run at 100 watt, set temperature at 50 C.
3. About 30 minute, when the temperature is reaching to 49 C, we have to heat shock DNA
sample that will load into the gel.
4. Use 94 C for heat shock in 3 minutes, then put immediately on ice that it ready for load
into gel.
5. Stop pre-run, clean the well and put the comb and deposit DNA sample 2.5 ul per well,
dont forget to deposit DNA marker on the first or the last well or any where.
6. Normally we run at 60 watt, 50 C about 16-cm length, it will take about 1.5 hour.
(Refer to figures below)

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Polyacrymide gel preparation

Wipe a gel plate Ad 700 ul of bind silane solution

Wipe a chamber Add the spacer

Set the gel chamber and gel plate Prepare acrylamide gel

Poor gel into the gel-set Push the comb

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Gel running

Pre-running Heat shock DNA sample

Put sample on ice immediately Clean the well

Put the comb Deposit DNA sample

Deposit DNA marker Run at 60 watt, 50 C

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Silver staining
1. Fix gel with 10% acetic acid (use 1 l), shake for 20 minutes in dark condition.
2. Wash 3 time with dH2O, 2 minute each.
3. Stain with silver-staining solution shake for 30 minutes.
Silver stain solution: 1 g/l of silver nitrate (AgNO3) + 1.5 ml/l of formaldehyde.
4. Quick wash for 10 seconds with dH2O.
5. Stain with developer-solution until DNA band are appear.
Developer-solution : 30 mg/l sodium carbonate anhydrous + 1 pellet of Sodium
thiosulfate + 1.5 ml/l of cold formaldehyde solution.
6. Stop of staining by adds 10% of acetic acid and shake.
7. Wash with dH2O for 5 minutes then let it dry.

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References

Aaij, C., Borst, P. The gel electrophoresis of DNA. Biochim. Biophys. Acta, 269:192, 1972

Fisher, M. P., Dingman, C. W. Role of molecular conformation in determining the

electrophoretic properties of polynucleotides in agarose-acrylamide composite gels.
Biochemistry, 10:895, 1971

Helling, R. B., Goodman, H. M., Boyer, H. W. Analysis of R. EcoRI fragments of DNA from
lamboid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol.,
14:1235, 1974

Maniatis, T., Jeffrey, A., and deSande, H.V. 1975. Chain length determination of small
double-and single-stranded DNA molecules by polyacrylamide gel electrophoresis.
Biochemistry 14:3787-3794.

Sambrook J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual.
2nd edition. Cold Spring Harbor Laboratory Press, USA.

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Sample of running of polyacrylamide gel from different PCR base methods.

AFLP: Amplified Fragment Length Polymorphism.

SSLP: Simple Sequence Length Polymorphism.

SSCP: Single Strand Conformation Polymorphism

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Reagents and solutions for agarose gel

Agarose gel (1% in 100ml buffer)

- weigh 1g of agarose
- add 100ml of buffer

50x Tris-acetate (TAE) buffer

- 242g Tris base
- 57.1 ml glacial acetic acid
- 100ml 0.5M EDTA (pH 8.0)
* dilute from 50x to prepare 1x buffer

5x Tris-borate (TBE) buffer

- 54g Tris base
- 27.5 g boric acid
- 20ml 0.5M EDTA (pH 8.0)

6x Gel loading buffers

- 0.25% bromphenol blue
- 0.25% xylene cyanol FF
- 30% glycerol in water
store at 4OC

Ethidium bromide
From a stock solution of 10mg/ml in water, add ethidium bromide to the gel with a
final concentration of 0.5ug/ml of the gel.

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Reagents and solutions for acrylamide gel

10x TBE buffer

- Tris Base 108 g.
- Boric Acid 55 g.
- 0.5M EDTA 20 ml.
- dH2O to 1000 ml.

10% Acetic acid (1 l)

900 ml of H2O + 100 ml of 100% acetic acid

40% Stock solution of polyacrylamide (1 l)

- Dissolve acrylamide 380 g. and bis-acrylamide 20 g. with dH2O 300 ml.
- Water to 1000 ml.

4.5 % Acrylamide (1 l)
- 5x TBE 200 ml.
- dH2O 350 ml.
- Urea 450 g.
- 40% acrylamide 112.5 ml.
- Water to 1000 ml.
- Filtering it.

10% APS (dH2O 10 ml + 1 g of APS)

Developer solution (1 l.)

- Na2CO3 30 g.
- Formaldehyde 1.5 ml.
- Na2S2O2 200 ul. (Soduim thiosulfate 10 mg/ml.)

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