Operating steps:

1, electrophoresis method

General nucleic acid detection only needs agarose gel electrophoresis; if you need high-resolution electrophoresis, especially only a few bp difference should be selected polyacrylamide gel electrophoresis; with the ordinary electrophoresis is not suitable for the huge DNA chain should be used pulse gel electrophoresis.

2. Gel concentration

For agarose gel electrophoresis, the concentration is usually between 0.5 and 2%, low concentration is used for electrophoresis of large nucleic acid fragments, and high concentration is used for analysis of small fragments. Low concentration gels are fragile and careful handling and use of good quality agarose is the solution.

3, buffer

Commonly used buffers are TAE and TBE, and TBE has better buffering capacity than TAE. The electrophoresis effect can be significantly improved by using the newly made buffer.

4, voltage and temperature

The electric field strength should not exceed 20V/cm and the electrophoresis temperature should be lower than 30℃, and for huge DNA electrophoresis, the temperature should be lower than 15℃.

5. Purity and status of DNA samples

Note that samples containing too much salt and impurity proteins can produce blurred bands and missing bands. Ethanol precipitation removes excess salt and phenol removes protein.

6. Sampling of DNA

The correct amount of DNA is necessary to ensure clear bands. Note that too much DNA sampling may result in blurred DNA banding, while too little DNA sampling results in weak or even missing banding signals.

7. Selection of Marker

DNA electrophoresis must use a DNA Marker or a positive control DNA of a known size to estimate the size of the DNA fragments, and the Marker should be densely packed near the size of the target fragments, so that the estimation of the target fragment size is more accurate.

8. Staining and observation of the gel

The commonly used nucleic acid stain in the laboratory is ethidium bromide (EB), which is effective in staining and easy to operate, but has poor stability and toxicity. Note that when observing the gel, the appropriate light source and excitation wavelength should be used according to the dye; if the excitation wavelength is not correct, the bands will not be easy to observe, and the phenomenon of blurred bands will occur.

2. Precautions:

1. Huge DNA strands may not be able to run out of the gel hole with ordinary electrophoresis, resulting in lack of bands.

2, high concentration of gel may make DNA bands of similar molecular size not easy to distinguish, resulting in the phenomenon of missing bands.

3. After the electrophoresis buffer has been used for many times, the ionic strength decreases, the pH increases, and the buffer performance decreases, which may cause the DNA electrophoresis to produce the phenomenon of fuzzy bands and irregular migration of DNA bands.

4. If the voltage and temperature are too high during electrophoresis, it may lead to the phenomenon of fuzzy bands and irregular DNA band migration. Especially, too much voltage may lead to small fragments running out of the gel and the phenomenon of missing bands.

5. Denatured DNA samples may result in blurred and missing bands and irregular DNA band migration. Do not heat the DNA sample before sampling, and dilution with 20 mM NaCl buffer can prevent DNA denaturation.

6. Too much DNA sample volume may result in blurred DNA banding, while too little DNA sample volume results in weak or even missing banding signals.

Extended information

The agarose gel has a network structure, and the molecules of the substance are resisted as they pass through, and large molecules of the substance are resisted when they are surging, so in gel electrophoresis, the separation of charged particles is not only dependent on the nature and amount of the net charge, but also on the size of the molecules, which greatly improves the resolving power.

But because their pore size is too large compared to proteins, their molecular sieving effect is negligible for most proteins, and they are now widely used in the study of nucleic acids.

Proteins and nucleic acids will have different charges depending on the pH, and will run at different speeds because of different forces in the electric field, and they can be separated according to this principle. The pH of electrophoresis buffer is between 6~9, and the ionic strength 0.02~0.05 is the most suitable. 1% agarose is commonly used as an electrophoretic support.

Agarose gel can distinguish DNA fragments with a difference of about 100bp, and its resolution is lower than that of polyacrylamide gel, but it is easy to prepare and has a wide separation range. Ordinary agarose gel separates DNA in the range of 0.2-20kb, and using pulsed electrophoresis, DNA fragments of up to 10^7bp can be separated.

There is a charge effect and a molecular sieve effect when DNA molecules swim in agarose gels.DNA molecules are negatively charged in pH solutions above the isoelectric point and move toward the positive pole in an electric field. Due to the structurally repetitive nature of the sugar-phosphate backbone, the same amount of double-stranded DNA has an almost equal net charge, so they can move toward the positive pole at the same rate.

Baidu Encyclopedia - Agarose Gel Electrophoresis