Download Frequently Asked Questions: Agarose Gel Electrophoresis

Agarose L03 (Cat.# 5003), TBE buffer products (Cat.#s T905, T9121, and T9122),
Tris-Acetate-EDTA Buffer (TAE) 50X Powder (Cat.# T9131),
Mupid® electrophoresis systems (Cat.#s AD110, AD160, and AD140)
Frequently Asked Questions: Agarose Gel Electrophoresis
Agarose gel electrophoresis is one of the most commonly used techniques in molecular biology for visualizing and isolating
nucleic acids during cloning projects, Southern or Northern blotting, etc. Despite the routine nature of agarose gel electrophoresis, an understanding of essential information about buffers, staining agents, and common problems is helpful to ensure
success.
Answers to frequently asked questions about agarose gel electrophoresis are presented here. For additional information, refer
to the technical literature and web pages for agarose gel electrophoresis products listed at the top of this FAQ.
Q1: What are the proper uses for agarose and acrylamide gels?
A1: Agarose gels are easy to prepare and can separate nucleic acids of a wide range of sizes. However, agarose gel electrophoresis is insufficient to detect small size differences between linear DNA molecules, even after optimization of gel thickness and
gel concentration.
Acrylamide gels are recommended for more precise resolution of DNA size, and can allow discernment between as little as
one base pair difference in size. Acrylamide gels can also be used for the electrophoresis of fragments ranging in size from a
few base pairs in length to a few kilobases. However, it is necessary to modify gel concentration according to the size range of
fragments being analyzed is necessary.
Q2: What are the differences between various types of agaroses?
A2: Less refined (inexpensive) agarose contains many impurities, which may inhibit the recovery of DNA from the agarose gel.
TAKARA Agarose L03 (Cat.# 5003) is purified, with contaminants carefully removed.
Q3: What is the best buffer to use for agarose gel electrophoresis?
A3: 1X TBE Buffer is recommended when the recovery rate for DNA less than 1 kb is not critical. DNA bands are sharper with
TBE Buffer compared with TAE Buffer. When approximate size estimation of DNA fragments is sufficient, TBE Buffer can provide
appropriately clear resolution of bands.
For DNA over 15 kb, 1X TAE Buffer promotes separation of DNA bands, and yields good results. However, during long electrophoresis runs, TAE Buffer requires periodic circulation and mixing at both the anode and cathode sides. The buffering capacity
declines depending on the voltage (v/h) and the volume of buffer chamber.
Regardless of the buffer used, the volume of buffer must be sufficient. We recommend a buffer level of 3 to 8 mm above the
top surface of the agarose gel. Insufficient buffer coverage increases the risk of gel drying. In contrast, flooding with excessive
buffer reduces the resistance between anode and cathode in the electrophoresis chamber, resulting in decreased voltage
gradient to the gel and decreased DNA mobility. In addition, the presence of excess buffer may also cause overheating of the
gel and smearing of DNA bands.
Q4: Which loading dye works best for loading DNA samples?
A4: Add xylene cyanol or bromophenol blue (BPB) to samples to monitor their migration of samples in the gel during electrophoresis. Dye mobility varies depending on the gel concentration, type of agarose, and electrophoretic buffer.
Q5: What gel staining agent is recommended?
A5: Ethidium bromide is widely used because it is inexpensive and allows visualization of DNA after electrophoresis using a
common UV illuminator. SYBR Green I stains double-stranded DNA; SYBR Green II stains RNA or single-stranded DNA; and
GelStar* stains single- or double-stranded DNA or RNA. However, maximum sensitivity detection requires special filters and
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light sources. In order of least sensitive to most sensitive, staining agents are: ethidium bromide < SYBR Green I < GelStar. Small
DNA fragments are inherently difficult to visualize after staining by any method, since the amount of DNA is small relative to
the mole quantity of fragments. GelStar and SYBR Green I offer highly sensitive detection, allowing the visualization of small
DNA fragments that cannot be seen with ethidium bromide staining.
Short wavelength ultraviolet (UV) irradiation induces DNA damage, such as breakage. If the purified DNA will be used for cloning, use an illuminator with a light source that emits long wavelength light and limit the duration of exposure to UV.
*Not available in all geographic locations. Check for availability in your region.
Q6: What is the difference between staining a gel after electrophoresis and placing dye in the electrophoresis chamber?
A6: Exposing a gel to staining agent after electrophoresis is complete results in even staining and provides clear visualization
of DNA fragments.In addition, because staining solution can be used multiple times, only a small amount of dye is required to
stain a large number of gels.
In comparison, staining agent may be added directly to the electrophoresis chamber during the process of gel preparation.
Although this procedure may not provide uniformly stained gels, it shortens the time required for staining and overall gel
handling time.
Compared with gels stained after electrophoresis is complete, adding SYBR Green to the gel lowers the sensitivity of detection
and results in less well-defined bands. Furthermore, adding SYBR Green to the gel itself is not recommended because the staining procedure may alter DNA mobility.
DNA mobility in gels containing GelStar* is slower compared with that in GelStar-free gels.
*Not available in all geographic locations. Check for availability in your region.
Q7: What are the advantages of using small vs. large electrophoresis devices?
A7: Small electrophoresis systems allow faster run times and are ideal for the analysis of plasmids and PCR products. Large
electrophoresis systems offer better resolving power and allow adjustments to voltage and current, making it easy to adjust the
duration of electrophoresis. In addition, the larger gel size can accommodate more samples.
Q8: What is the proper way to dispose of staining solutions?
A8: Follow all applicable safety and disposal regulations at your institution. Since most solutions used to stain nucleic acids
contain mutagenic compounds, do not pour untreated stain solutions down the drain. Pass waste stain solutions through a
commercially available adsorption column (such as bondEx Starter Kit, Cat. # 740701*) or activated charcoal prior to disposal.
*Not available in all geographic locations. Check for availability in your region.
Troubleshooting
T1: I was unable to load samples into the wells of an agarose gel successfully.
A1: DNA solutions on their own have low specific gravity and are difficult to load into wells.
Takara’s restriction enzymes come with 10X Loading Buffer (containing an electrophoresis dye and glycerol) for sample loading.
Add 1/10 volume loading buffer to samples and mix well. Because this buffer contains glycerol as a density agent, samples will
slowly settle into the wells.
T2: After electrophoresis of a restriction enzyme-digested plasmid, there was a strongly stained diffuse
band of several hundred base pairs. What is this?
A2: Without RNase treatment during DNA preparation, RNA (primarily rRNA) that has been extracted along with the plasmid
will show up as a very strongly stained diffuse band with apparent size on the gel of several hundred base pairs. This will not
affect transformation into E. coli, restriction enzyme treatment, or PCR. To avoid this artifact, add RNase A to the loading buffer
or treat the plasmid DNA sample with RNase A.
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T3: The loading dye migrated in an arc pattern during electrophoresis.
A3: High voltage electrophoresis creates an uneven temperature throughout the gel, leading to different electrophoretic
mobility rates between the edges versus the center of the gel. To avoid this phenomenon, perform cool the electrophoresis
system during the run or reduce the voltage.
T4: The bands were blurry in images of the gel taken after electrophoresis.
A4: Air bubbles trapped in wells or incomplete immersion of wells in buffer will result in blurry bands after electrophoresis.
After loading samples, please make sure that wells are completely immersed in buffer.
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