Agarose Gel Electrophoresis
What is Agarose Gel Electrophoresis?
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA,
or RNA molecules by size however proteins can also be separated on agarose gels. Separation of the
molecules is achieved by moving negatively charged nucleic acid molecules through an agarose matrix
in an electric field (see electrophoresis). Shorter molecules move faster and migrate further than longer
ones.
Applications of Agarose Gel Electrophoresis
Agarose gel electrophoresis is mainly used in analysis or separation of DNA and RNA molecules
(although proteins can also be separated on agarose gels), however separation also allows the
purification of specific sizes of DNAs after restriction enzyme digestion. This is usually used in cloning to
obtain cut plasmids, in which agarose gel electrophoresis separates cut vectors from uncut ones.
Thus agarose gels allow:
1.
Separation of restriction enzyme digested DNA including genomic DNA, prior to Southern Blot
transfer. It is also often used for separating RNA prior to Northern transfer.
2.
Analysis of PCR products after polymerase chain reaction to assess for target DNA
amplification.
3.
Allows for the estimation of the size of DNA molecules using a DNA marker or ladder which
contains DNA fragments of various known sizes.
4.
Allows the rough estimation of DNA quantity and quality.
5.
Quantity is assessed using lambda DNA ladder which contains specific amounts of DNA in
different bands.
6.
Quality of DNA is assessed by observing the absence of streaking or fragments (or
contaminating DNA bands).
7.
Other techniques rely on agarose gel electrophoresis for DNA separation including DNA
fingerprinting.
Advantages and Disadvantages of Agarose Gel Electrophoresis
The advantages are that the gel is 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
different forms of genetic material may run in unpredictable forms.
Migration in Agarose Gels
The most important factor is the length of the DNA molecule, smaller molecules travel farther. But
conformation of the DNA molecule is also a factor. To avoid this problem linear molecules are usually
separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs.
Increasing the agarose concentration of a gel reduces the migration speed and enables separation of
smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by
the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution
(above about 5 to 8 V/cm).[citation needed]
Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different
speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid.
Resolving Limits of Agarose Gels
Agarose gels can be used for the separation of DNA fragments ranging from 50 base pairs to several
megabases (millions of bases) by electrophoresis. More commonly however, agarose gel
electrophoresis is used to separate DNA from PCR and cloning in the range of 100bp to about 15kb.
Run times are about 30 minutes - 1 hour long.
Small DNAs or RNAs (smaller than 100bp) are better separated by polyacrylamide gels, however 2-3%
agarose gels may be adequate to separate even 50bp fragments from much larger nucleic acids.
Recently however, it has been shown that up to one base pair size difference could be resolved on a 3%
agarose gel with an extremely low conductivity medium such as 1 mM Lithium borate (Brody JR,
Calhoun ES, Gallmeier E, Creavalle TD, Kern SE (2004). Ultra-fast high-resolution agarose
electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques. 37:598-602.).
Table of Agarose Gel Percentage and Efficient Range of Separation:
As you can see low percentage agarose gels are best for the separation of large DNA molecules,
whereas higher percentage gels are best for smaller DNAs.
DNA Purification with Agarose Gel Extraction
As mentioned before, agarose gel electrophoresis allows the separation and thus the purification of DNA
for cloning. However, this usually requires high purity low melt agarose if the DNA is to be extracted
from the gel.
Agarose Gel Electrophoresis Buffers
Depending on the size of the DNA electrophoresed and the application, different buffers can be used for
agarose electrophoresis. TAE buffer (or Tris Acetate EDTA) is the most common used agarose gel
electrophoresis buffer. TAE has the lowest buffering capacity of the buffers, however TAE offers the
best resolution for larger DNA. However, TAE requires a lower voltage and more time.
However TBE buffer (Tris/Borate/EDTA) is often used for smaller DNA fragments (ie less than 500bp).
Sodium borate or SB buffer is a new buffer, however it is ineffective for resolving fragments larger than 5
kb. However SB has advantages in its low conductivity, allowing higher voltages (up to 35 V/cm). This
could allow a shorter analysis time for routine electrophoresis.
Reference:
http://www.molecularstation.com/agarose-gel-electrophoresis/
Agarose Gel Preparation
In your first run you may run only your standards and once you
confirmed that your system is operating correctly you may run your
samples together with the standards.
Now you are ready to start preparing the gel.
1) Seal the open borders of a clean and dry glass plate with tape for
forming a mold.
2) Prepare an adequate volume of electrophoresis buffer (TBE:
Tris/borate/EDTA or TAE: Tris/acetate/EDTA) to fill the electrophoresis
tank and to prepare the gel.
3) Add the needed amount of powdered agarose (typically 8-15%) ( we
prepare 1% - e.g. 1 gram of agarose in 100 ml of TAE buffer ) to the
electrophoresis buffer (TBE or TAE) in an Erlenmeyer flask. The buffer
should not occupy more than 50% of the volume of the flask.
4) Heat the agarose solution in a microwave oven or in water bath to
allow all of the grains of agarose to dissolve. If part of the buffer
evaporated during the heating, bring the solution back to the original
volume through the addition of buffer.
5) Cool down the solution to 60 C. You may add ethidium bromide (Et Br
it is dangerous carcinogenesis chemical)) ( or Gel stare dye which is not
dangerous and safer) , what we used it)(10 mg/l in water to a final
concentration of 0.5 um/ml) ( Note : we used 3-4 ul of EtBr) in this step
or you can submerge the gel in an Et Br solution once it solidifies.
6) Position the comb 0.5-1.0 mm above the plate, thus permitting that a
complete well is formed when the agarose solidifies. Air bubbles under or
between the teeth of the comb should be avoided. Using a Pasteur pipette
seal the glass plate with small amounts of agarose solution. Once the seals
are set, pour the gel in the glass plate.
7) Remove the comb and the tape when the gel has completely hardened
(30 to 40 minutes at room temperature) and place the gel in the
electrophoresis tank. Add enough electrophoresis buffer to the tank to
cover the gel (about 1 mm of depth). The top of the wells should be
submerged.
8) Mix the samples and standards with appropriate amounts of loading
buffer (10x) ( We can use orange dye or Bromophenole blue dye). Slowly
load the mixture into the wells with a micropipette. Avoid any mix of the
samples between wells.
9) Close the lid of the tank and be sure that the samples are placed at
the correctly positioned with respect to the anode and the cathode (DNA
will migrate toward the anode). Apply the desired voltage (1-5 V/cm) to
the gel to begin the electrophoresis. Endurance and voltage of the
electrophoresis depends on the nature of the sample run and the desired
resolution. ( We put it at 250 volt for approximately 15-20 min.)
10) If the unit is working you should see bubbles that are formed in the
buffer due to the electrical field that has been creates; and later you
should see the dyes running in the gel.
11)If ethidium bromide was present in the agarose gel, DNA may be
photographed by with UV light (>500uW/cm2). The location of DNA
within the gel and may be determined by a film image of light transmitted
by a fluorescing DNA. Thus, a wide range of bands or fragments of DNA
can be detected on the film (from 200 bp to ~ 50 kb in length). Size and
amount of DNA can be calculated running standards of known size and
concentration. DNA bands may be recovered from the gel and utilized
for a diversity of cloning designs. See Current Protocols on Molecular
Biology (1995, John Wiley & Sons Inc., Virginia Benson, Canada) for
more information on cloning and DNA analysis protocols.
Visualization of DNA bands with UV light