Gel electrophoresis
Principle of gel analysis
Gels allow separation and identification of nucleic acids based on charge migration.
Migration of nucleic acid molecules in an electric field is determined by size and conformation,
allowing nucleic acids of different sizes to be separated. However, the relationship between the
fragment size and rate of migration is non-linear, since larger fragments have greater frictional
drag and are less efficient at migrating through the polymer. Agarose gel analysis is the most
commonly used method for analyzing DNA fragments between 0.1 and 25 kb.
Concentration of agarose used for separating DNA of different sizes
Agarose concentration (% w/v)
DNA fragment range (kb)
1.0
0.5–10
1.5
0.2–3
2.0
0.05–2
Electrophoresis buffers
The most commonly used buffers for agarose gel electrophoresis are TBE (Tris-borate–EDTA)
and TAE (Tris-acetate–EDTA). TAE has a lower buffering capacity than TBE and is more easily
exhausted during extended electrophoresis. TBE gives better resolution and sharper bands, and is
particularly recommended for analyzing fragments <1 kb. The drawback of TBE is that the borate
ions in the buffer form complexes with the cis-diol groups of sugar monomers and polymers,
making it difficult to extract DNA fragments from TBE gels.
Pouring the gel
1. Prepare enough 1x running buffer both to pour the gel and fill the electrophoresis tank.
2. Add an appropriate amount of agarose (depending on the concentration required) to an
appropriate volume of running buffer (depending on the volume of the gel tray being used) in a
flask or bottle.
3. Heat the slurry in a microwave or boiling water bath, swirling the vessel occasionally, until the
agarose is dissolved.
(Ensure that the lid of the flask is loose to avoid buildup of pressure. Be careful not to let the
agarose solution boil over as it becomes superheated)
4. Cool the agarose to 55–60°C.
5. Pour the agarose solution onto the gel tray to a thickness of 3–5 mm. Insert the comb either
before or immediately after pouring. Leave the gel to set (30–40 min).
(Ensure that there is enough space between the bottom of the comb and the glass plate (0.5–1.0
mm) to allow proper formation of the wells and avoid sample leakage.
6. In the meanwhile, prepare your samples: 5 µl PCR product + 1 µl loading buffer
7. Carefully remove the comb and adhesive tape (if used) from the gel. Fill the tank containing
the gel with electrophoresis buffer.
8. Add enough buffer to cover the gel with a depth of approximately 1 mm liquid above the
surface of the gel. If too much buffer is used the electric current will flow through the buffer
instead of the gel.
9. Apply samples in gel loading buffer to the wells of the gel.
Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of
the wells.
10. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode).
Electrophoresis apparatus should always be covered to protect against electric shocks.
11. Turn on the power supply and run the gel at 80 V for 45 minutes.
Avoid use of very high voltages which can cause trailing and smearing of DNA bands in the gel,
particularly with high-molecular-weight DNA.
12. Monitor the temperature of the buffer periodically during the run. If the buffer becomes
heated, reduce the voltage.
Melting of an agarose gel during electrophoresis is a sign that the voltage is too high, that the
buffer may have been incorrectly prepared or has become exhausted during the run.
Ethidium bromide staining after electrophoresis
12. Soak the gel in (10 µl ethidium bromide (10mg/ml) in 500ml H2O) solution of ethidium
bromide (in water or electrophoresis buffer) for 30–40 minutes.
13. Rinse the gel with buffer or water before examining it to remove excess ethidium bromide.
Staining buffer can be saved and re-used.
Hazard
Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and take appropriate
safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not
provide full protection.
Visualization
Ethidium bromide–DNA complexes display increased fluorescence compared to the dye in
solution. This means that illumination of a stained gel under UV light (254–366 nm) allows bands
of DNA to be visualized against a background of unbound dye. The gel image can be recorded by
taking a Polaroid™ photograph or using a gel documentation system.
Precaution
UV light can damage the eyes and skin. Always wear suitable eye and face protection when
working with a UV light source. UV light damages DNA. If DNA fragments are to be extracted
from the gel, use a lower intensity UV source if possible and minimize exposure of the DNA to
the UV light.
Disposal of Ethidium-bromide waste
14. All items that were in contact with EthBr must be disposed of in the designated waste
container (marked with "Ethidium bromide waste") within the Gel-Doc-Area, including gels,
tissue paper to clean UV table, and Nitril-gloves
PAGE: Poly-Acrylamid-Gel-Electrophoresis
Pouring of PAGE-gels
1) set-up of gel-plates in gel-caster as illustrated in manufacturer's instructions
2) premix 10 ml Acrylamid-Gel-solution, using 5ml-Pipet (sufficient for 2 mini-gels):
Acrylamide concentration
10.5%
5.1%
5.4 ml 7.2 ml ds H2O
3.5 ml 1.7 ml 30% Acrylamide solution
1 ml
1 ml
10X TBE-buffer
20 µl
20 µl TEMED
70 µl
70 µl 10% APS
(Add APS directly before pouring the gel!)
3) pour gel using 5ml-pippette up to the top of the short plate (Avoid bubbles!)
4) insert comb
5) leave for 1h to polymerise
Preparation of samples
6) mix 2 µl of bromo-phenol-blue (BPB) loading buffer and 1-5 µl of sample DNA
7) similarly prepare 5 µl pGEM marker DNA (see below)
Gel-Electrophoresis
8) set-up gel in gel-tank as illustrated in manufacturer's instructions
9) add 130 ml 1X TBE to inner chamber & 200 ml 1X TBE to outer chamber of gel-tank
10) slowly remove comb
11) thoroughly rinse sample wells using a syringe
12) load samples & close lid of gel-tank
13) run the gel for 1h (45 min) at 80V
14) switch off power & remove gel-cassettes as illustrated in manufacturer's instructions
Staining of gel
17) place gel cassette in water bath & carefully remove short plate
18) Stain PAGE-gel on large plate in SYBR-green (1:10,000 SYBR Green in 100 ml H2O) for 1 h
(30 min if solution is fresh)
19) transfer PAGE-gel from large plate onto the UV-table using fish slice
20) check results + take photograph
Note: Always wear Nitril gloves while working with SYBR-Green
Disposal of SYBR-Green waste
21) All items that were in contact with SYBR Green must be disposed of in the designated waste
container (marked with "SYBR Green waste") within the Gel-Doc-Area, including gels, tissue
paper to clean UV table, and Nitril-gloves
Marker DNA for Agarose Gel Electrophoresis
λ-DNA cut with HindIII [λ
λ-HindIII]
size of bands [kb]
23.130
9.400
6.560
4.360
2.300
2.030
0.564
0.125
weight of bands in [ng] (per 100ng loaded)
50.0
20.0
13.5
10.0
5.0
5.0
1.2
0.3
λ-DNA cut with HindIII, EcoRI, BamHI
[λ
λ-BEH]
pGEM-marker
[PGM]
size of bands [kb]
16.270
8.950
5.540
3.680
3.410
2.790
2.580
2.470
2.450
1.980
1.570
1.320
1.130
0.930
0.840
0.780
0.560
0.490
0.140
size of bands [kb]
2.645
1.605
1.198
0.676
0.517
0.460
0.396
0.350
0.222
0.179
0.126
0.075
0.065
0.051
0.036