Sample Preparation and Loading
Due to the use of a continuous buffer system without stacking gel in most cases, the samples should have an appropriate concentration and a small volume. Use a pipette to slowly add the sample, with 5-10 μg per well, to avoid a significant decrease in resolution. When loading sample, the power supply must be turned off. The sample should contain an indicator dye (0.025% bromophenol blue or orange) and sucrose (10-15%) or glycerol (5-10%) to increase its density, concentrate the sample, and prevent diffusion. However, sometimes sucrose or glycerol may cause U-shaped bands in electrophoresis results, which can be avoided by using 2.5% Ficoll (polyvinylpyrrolidone).
Electrophoresis
The voltage for electrophoresis is 5-15 V/cm, generally around 10 V/cm. For the separation of large molecules, the voltage should be lower, usually not exceeding 5 V/cm.
Staining
Fluorescent dye ethidium bromide (EB) is commonly used for staining to observe DNA bands in agarose gel. EB can insert between base pairs of DNA molecules, causing EB to bind with DNA. The absorption of 260 nm ultraviolet light by DNA is transferred to EB, and the bound EB emits fluorescence at 590 nm in the red-orange region of the visible light spectrum. Staining the gel in a 1 mmol/L MgSO4 solution for 1 hour can reduce background fluorescence caused by unbound EB, facilitating the detection of small amounts of DNA.
EB dye has several advantages: it is easy to use, does not break nucleic acids, has high sensitivity, and can stain both DNA and RNA. EB can be added to the sample and tracked using UV absorption at any time. After staining, EB can be removed by extraction with n-butanol.
However, EB dye is a potent mutagen, and precautions, such as wearing polyethylene gloves, should be taken during handling. Acridine orange is also a commonly used dye because it can differentiate between single-stranded and double-stranded nucleic acids (DNA, RNA). It exhibits green fluorescence (530 nm) for double-stranded nucleic acids and red fluorescence (640 nm) for single-stranded nucleic acids. Additionally, other dyes such as methylene blue, methylene green, and quinoline B can be used for staining.
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Post time: Dec-20-2023