Detection of DNA in agarose gels
Nucleic acids in agarose gels can be detected by staining under UV light at a wavelength of 300 nm. Two methods for staining nucleic acids in agarose gels are introduced: ethidium bromide (EB) staining and SYBR Gold staining. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Detection of DNA in agarose gels
Principle
Nucleic acids in agarose gels can be detected by staining under UV light at a wavelength of 300 nm. Two methods for staining nucleic acids in agarose gels are described: ethidium bromide (EB ) staining and SYBR Gold staining.
Materials and Instruments
Ethidium bromide SYBR Gold Move 1. Gel ethidium bromide staining method For more product details, please visit Aladdin Scientific website.
The easiest and most common way to visualize DNA in agarose gels is by staining with the fluorescent dye ethidium bromide (Sharp et al. 1973). Ethidium bromide contains a tricyclic planar group that can be embedded between the stacked bases of DNA. It binds to DNA with little base sequence specificity. In saturated solutions of high ionic strength, one ethidium bromide molecule is inserted approximately every 2.5 bases (Waring 1965). When a dye molecule is inserted, its planar group is perpendicular to the axis of the helix and interacts with the upper and lower bases by van der Waals forces. The fixed position of this group and its close proximity to the bases causes the DNA-bound dye to fluoresce with an increased fluorescence yield compared to that of the dye in free solution.The DNA absorbs UV radiation at 254 nm and passes it on to the dye, while the bound dye itself absorbs light radiation at 302 nm and 366 nm. In both cases, the absorbed energy is re-emitted at 590 nm in the red-orange region of the visible spectrum (LePecq and Faoletti 1967). Since the fluorescence yield of the ethidium bromide-DNA complex is 20-30 times higher than that of the dye without bound DNA, DNA bands as small as 10 ng can be detected when the gel contains free ethidium bromide (0.5 μg/ml). 
Ethidium bromide can be used to detect either single-stranded or double-stranded nucleic acids (DNA or RNA). However, the affinity of the dye for single-stranded nucleic acids is relatively low, so its fluorescence yield is also relatively low. In fact, most of the fluorescence of single-stranded DNA or RNA staining is produced by the binding of the dye to the molecule to form a short intrastrand double helix.
Ethidium bromide is usually prepared in a 10 mg/ml storage solution with water and stored at room temperature in a brown bottle or a bottle wrapped in aluminum foil. The dye is usually incorporated into agarose gels and buffers at a concentration of 0.5 μg/ml. Note: Ethidium bromide should not be incorporated into polyacrylamide gel preparations due to its ability to inhibit acrylamide polymerization. Acrylamide electrophoresis is usually stained with ethidium bromide at the end of electrophoresis.
Although the electrophoretic mobility of linear DNA is reduced by about 15% in the presence of this dye, the greatest advantage is that it can be detected directly under UV light during or after electrophoresis. When there is no EB in the gel, the DNA bands in the gel are much clearer. Therefore, when it is necessary to know the exact size of the DNA fragments (e.g., for identification of DNA restriction enzyme profiles), the gel should be electrophoresed without EB and stained with EB at the end of electrophoresis. For staining, the gel should be immersed in electrophoresis buffer containing EB (0.5 μg/ml) and stained at room temperature for 30-45 min. After staining, there is usually no need for destaining. However, when detecting small amounts of DNA (<10 ng), the stained gel should be immersed in water or 1 mmol/L MgSO4 and decolorized for 20 min at room temperature for easier observation.
2. Gel SYBR Gold staining
SYBR Gold is the trade name of a new type of extremely sensitive dye. SYBR Gold-DNA complex generates much more photons than EB-DNA complex, and the intensity of the excited fluorescence signal is more than 1000 times of that of EB-DNA complex. Therefore, SYBR Gold staining can detect less than 20 pg of double-stranded DNA in agarose gels (1/25 of the minimum detectable amount of EB staining). In addition, a single-stranded DNA with a band as small as 100 pg or 300 pg of RNA can be detected with SYBR Gold staining.The maximum excitation wavelength of the SYBR Gold dye is 495 nm, and there is a second excitation peak at 300 nm. The maximum excitation wavelength of SYBR Gold dye is 495 nm, with a second peak at 300 nm, and it emits fluorescence at 537 nm. 
After separation of the DNA fragments by gel electrophoresis, the gel is immersed in a dilution of the SYBR Gold stock solution (1:10,000). SYBR Gold should not be added to melted gels or to gels prior to electrophoresis because the electrophoretic bands of nucleic acids in the gel are severely distorted in the presence of SYBR Gold.
The highest sensitivity is achieved when the gel stained with SYBR Gold is exposed to UV irradiation at a wavelength of 300 nm. It can be illuminated by means of a green or yellow filter as described later. Since SYBR Gold is sensitive to fluorescence, the working solution (1:10,000 dilution of the storage solution) should be freshly prepared on the same day in electrophoresis buffer and stored at room temperature.
3. Imaging of DNA in gels
EB-stained gels can be imaged with transmitted or incident UV light. Most commercially available UV sources emit light at 302 nm, and at this wavelength, the fluorescence yield of the EB-DNA complex is much higher than at 366 nm and slightly lower than at 254 nm. At 302 nm, DNA is nicked to a much lesser extent than at 254 nm (Brunk and Simpson 1977).
It is now possible to examine EB-stained gels with a complete imaging system. This system consists primarily of a light source, a fixed-focus digital camera, and a thermal printer. The CCD camera in these systems utilizes a wide-angle zoom lens (75 mm focal length). This camera can detect trace amounts of EB-stained DNA (claims of 0.01-0.5 ng). In more advanced imaging systems, the gel image can be exported directly to a computer for direct visualization. The image can be processed on the computer monitor prior to printing in a variety of ways, including field of view, focal length, and cumulative exposure time. Each image can be printed and stored as a file in various forms, and software can be used to further process the image. The file size of an agarose image is approximately 0.3 M bytes. Therefore, to save a large number of images, a system with a large storage capacity is required. While printing individual images costs only a few cents and imaging via Polaroid costs about $1, a small imaging system can cost several thousand dollars, plus a host of other accessories, and VENDORS sells a number of gel imaging systems, notably: AlpHa Innotech (San Leandro, California), Fotodyne (Hartland, California), and Fotodyne (Hartland, California), AlpHa Innotech (San Leandro, California), Fotodyne (Hartland, Wisconsin) and Stratagene (La Jolla, California). 
Although results can be analyzed in a timely manner from images obtained by gel imaging systems, printed images tend to fade and lack layers during storage. Therefore, to obtain more satisfactory images with a longer retention time, highly sensitive Polaroid film types 57 or 667 (ASA 3000) should be used. With a high-efficiency UV light source (>2500 mW/cm2 ), a Wratten 22A (red or orange-red) filter, and a good lens (f=135 mm), stripes of as little as 10 ng of DNA can be obtained after a few seconds of exposure. With longer exposure times and a strong UV light source, fluorescence from as little as 1 ng of DNA can be recorded on film. Trace DNA bands can be detected with conventional wet-processed films (e.g., Kodak No. 4155) and lenses with smaller focal lengths (f=75 mm). In this case, the lens is closer to the gel and the image is concentrated in a smaller area of the film. This also allows more flexibility in developing and printing images.
By using SYBR Gold (Molecular Probe) to stain the DNA, the sensitivity of the image can be increased by a factor of 10 to 20. Of course, this increase in sensitivity comes with a steep increase in cost, with 10L of SYBR Gold working solution costing more than $100, while the same amount of EB costs about 5 cents. Simultaneous detection of SYBR Gold-stained gels requires a camera with yellow or green gelatin or cellophane filters (S-7569, Molecular or Kodak) under UV transmitted light at a wavelength of 300 nm.