Agarose Gel Electrophoresis

Section I

Say goodbye to your DNA samples—Mistakes in agarose gel electrophoresis

1. Use water instead of buffer for the gel or running buffer

Agarose gels are cast and run using TAE or TBE buffer. If you do use water, your gel will melt shortly after applying voltage to the electrophoresis unit. TAE, TBE, and water are all clear solution; therefore, check the label on the container during setup.

2. Use the wrong percentage of agarose

The standard percentage of agarose used to run a DNA gel is usually around 1.0%. A higher agarose percentage enhances resolution of smaller bands; conversely, a lower agarose percentage gives better resolution and separation of higher molecular-weight bands. If the wrong percentage is used, it can be difficult to visualize the DNA bands reliably. Be cautious when using low-percentage agarose gels; they are typically weak and more prone to breakage.

3. Switch the electrophoresis leads to the power supply

It is an easy mistake, but rather frustrating if you accidentally switch the electrophoresis unit leads to the power supply. The samples will run backwards, and because the wells are so close to the end of the gel, you will likely lose all the samples. Ensure the DNA samples enter the gel in the right direction after starting your run; switch the leads if you see your bands heading in the wrong direction.

Section II

5 ways to achieve higher resolution in agarose gels

1. What is the right buffer for your agarose gel electrophoresis?

The type of buffer used to run DNA in agarose gel electrophoresis depends primarily on the size of DNA fragment and the post-electrophoresis application. The two most popular types of buffers for running agarose gels are Tris-acetate with EDTA (TAE) and Tris-borate with EDTA (TBE). Because both buffers have a near-neutral pH, the DNA in the buffers has a net negative charge and migrates toward the anode (+) end of the gel apparatus.

For small DNA (<1000 bp), and if there is no plan to extract the DNA, then 1x TBE buffer is recommended. TBE buffer has a high ionic strength and buffering capacity. TAE buffer, together with low field strength (1–2 V/cm), is preferred for separating large DNA (12–15 kb). TAE buffer interacts with agarose, resulting in lower electroendosmosis, larger apparent pore size, and lower field strength compared to agarose gels in TBE buffer, all of which decrease the tendency of large DNA to smear.

2. How much DNA should you load to get the best resolution?

The amount of DNA sample can be varied, what is important is the quantity of DNA in the band(s) that you are separating. The minimal quantity of DNA that may be detected is dependent on the stains used, e.g., 3 ng of DNA in a band can be detected using SYBR Safe DNA gel stain. The maximum quantity of DNA in a band that is still clear and well-defined is approximately 100 ng.

3. How does gel casting affect the band resolution?

The recommended thickness for agarose gel is 3–4 mm; a gel thicker than 5 mm will result in fuzzy bands and higher staining background. Similarly, the amount of running buffer to cover over the gel in an electrophoresis apparatus is 3–5 mm. Too much buffer will decrease DNA mobility and cause band distortion.

The thickness of the comb is also important and significantly affects resolution. A thin comb (1 mm) gives very well-defined bands, while a thick comb gives thick bands leading to reduced resolution. The comb should be well cleaned to eliminate possible residues that could cause wavy lines in the lanes. In addition, add buffer before removing the comb to minimize tearing part of the agarose near the well.

4. Does the type of gel affect band resolution?

Depending on the application and size of the DNA, the type of agarose used can affect the DNA resolution. Gel strength, gel melting temperature, and electroendosmosis are important factors when choosing the right agarose. Invitrogen offers a range of agarose types that are specifically designed to optimize results for particular fragment sizes and applications.

  • UltraPure Agarose is standard melting temperature, multi-purpose agarose designed for routine separation analysis. UltraPure Agarose resolves DNA and RNA fragments in the 500 bp to 23,000 bp range.
  • UltraPure Agarose 1000 is a specialized agarose that provides higher resolution of PCR fragments and other short DNA fragments. Agarose 1000 is easier to handle because it has a stronger gel structure: gel strength is more than 1,400 g/cm2.
  • For low melting/gelling temperature agarose, UltraPure Low Melting Point Agarose is recommended for rapid DNA gel extraction protocols. The resolution range for this agarose is 50 bp to 1,000 bp.

5. How do you choose the running conditions for agarose gel electrophoresis?

The recommended voltage is 4–10 V/cm (distance between anode and cathode, not the length of the gel) in the gel electrophoresis unit. If the voltage is too low, then the mobility is reduced and band broadening will occur due to diffusion. If the voltage is too high, the band resolution is reduced, mainly because of gel overheating.