DNA and RNA Gel Documentation with the Odyssey Imagers

Nucleic Acid Electrophoresis

Nucleic acid electrophoresis is a common laboratory technique that separates nucleic acids based on molecular size and structure through an agarose gel with the aid of an electric current.

General Workflow

The workflow for nucleic acid electrophoresis can differ based on the type of gel and electrophoresis apparatus. This workflow serves as a generalized example when using a hand-poured gel and electrophoresis chamber.

To begin, an electrophoresis chamber is assembled, and the running buffer added. The buffer is critical for allowing the electric current to move through the gel and stimulate the molecular migration. The agarose gel is submerged in the chamber, and the stained or unstained nucleic acid samples are loaded on the gel with a marker with bands of known sizes. Once the run is complete, bands are stained as needed, and the gel is imaged. The imaged bands are then compared to the marker to determine their relative sizes.

Types of Stains

There are a wide variety of commercial stains for imaging the results of nucleic acid electrophoresis. Several common stains include Syto® 60 Nucleic Acid Stain, Ethidium Bromide (EtBr), SYBR® Safe DNA stain, and GelRed® Nucleic Acid Stain. When selecting the appropriate one for an experiment, always verify that its excitation and emission suit your needs and imager (Table 1). Always consider both the stain and imager together when making your selection, since stain selection can depend on or influence the type of imaging system used. The Odyssey® Family Imagers and the D-DiGit® Gel Scanner, for example, are designed to image specific stains.

Table 1. Common dye compatibility with Odyssey Imagers and D-DiGit Gel Scanner.

EtBr SYBR Safe DNA Gel Stain Syto 60 Nucleic Acid Stain GelRed Nucleic Acid Stain Pro-Q Diamond Phosphoprotein Gel Stain GelGreen® Nucleic Acid Gel Stain MIDORI Green Advance Nucleic Acid Stain GreenView DNA Gel Stain
Odyssey M Imager checkmark checkmark checkmark checkmark checkmark checkmark checkmark checkmark
Odyssey XF Imager checkmark checkmark checkmark checkmark checkmark checkmark checkmark
Odyssey Fc Imager checkmark checkmark checkmark checkmark checkmark checkmark checkmark
Odyssey DLx Imager checkmark
Odyssey CLx Imager checkmark
D-DiGit Gel Scanner checkmark checkmark checkmark checkmark checkmark

Detection Methods

Nucleic acid detection involves comparing nucleic acid bands with known band markers to determine molecular sizes. The necessary detection method can be determined by the type and number of stains (Table 1).

A detection and imaging protocol is available in the Imaging Nucleic Acid Gels on Odyssey® Imagers and D-DiGit® Gel Scanner Application Guide.

Imager Considerations

When preparing to detect or image nucleic acid gels, a key step is selecting the appropriate imager. Always consider its versatility; a suitable imager matches the fluctuation and growth of your research initiatives over time, saving costs on replacement or additional imagers. Two imagers and scanners that can image a variety of stains include the Odyssey M Imager and the D-DiGit Gel Scanner. The Odyssey M Imager quickly images commonly used stains without risk of saturation (Figure 1); it also accommodates membranes, plate-based assays, slides, and protein gels—making it a highly versatile option. In addition to imaging various stains, the D-DiGit Gel Scanner protects against potentially harmful ultraviolet light and EtBr; it also allows for DNA band extraction using a viewing shield and suits large and small gels.

figure 1
Figure 1. Dilutions (250 to 2 ng) of a 1 kb ladder were loaded on a 1% agarose gel. Gels were post-stained with A) 1:10,000 dilution of SYBR® Safe in 1X TAE buffer for 40 minutes, B) 3X GelRed stain in H2O for 30-45 minutes, and C) Ethidium Bromide for 30-45 minutes. Images were collected on the Odyssey M imager in the A) 530 and B, C) 590 channels.
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Figure 2. DNA gels imaged on the Odyssey Fc using Ethidium Bromide, SYBR Safe and Syto 60. The EtBr gel was also documented using Polaroid film to show the comparison.
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Figure 3. DNA Gel imaged on the D-DiGit DNA Gel Scanner using SYBR Safe. A 1:2 dilution series of Lambda DNA – BstEII Digest was run on 0.8% agarose gels containing 1X TAE buffer. Lane 1 contains 250 ng of DNA; sample loading volumes decreased progressively down to 2 ng in Lane 8. The gel was stained post-electrophoresis with 1X SYBR Safe (Invitrogen) and imaged on the D-DiGit Gel Scanner using a 6X exposure setting.

A second key factor to consider when selecting an imager is its efficiency and precision. The ideal imager offers high resolution for unsaturated, high-quality images. While a broad dynamic range of six logs or more is a key feature for all Odyssey Imagers, each one offers unique benefits. The Odyssey M Imager, for instance, uses up to 5 µm resolution for precise, detailed imaging and is compatible with numerous nucleic acid stains. The Odyssey XF Imager can image membranes and gels and is compatible with several nucleic acid stains. Meanwhile, the Odyssey DLx Imager scans up to six microplates, nine mini-blots, or thirty slides simultaneously and can image Syto 60. Imagers like these should be used to capture high-resolution images without saturation and move your research forward.