Notes are as follows:
1. Buffer system: When there are no ions, the conductivity is minimum, and DNA does not migrate, or migrates extremely slowly, in a buffer with high ionic strength. , the conductivity is very high and heat is generated, which may cause DNA denaturation, so attention should be paid to whether the buffer is used correctly. During long-term high-voltage electrophoresis, it is advisable to frequently refresh the buffer or circulate the buffer between the two tanks.
2. Agarose: Agarose from different manufacturers and batch numbers has different impurity contents, which affects the migration of DNA and the intensity of the fluorescence background, and should be used selectively.
3. Gel preparation: The buffer added to the gel should be consistent with that in the electrophoresis tank. The dissolved gel should be poured into the plate in time to avoid solidification before pouring. The gel poured into the plate should avoid bubbles, which may affect the electrophoresis results.
4. Sample addition amount: Under normal circumstances, a 0.5cm wide comb can add 0.5μg of DNA. The amount of sample addition depends on the size of the sample well and the number and size of the fragments in the DNA. It is determined that too much amount will overload the sample well, resulting in tailing and dispersion. This phenomenon is more obvious for larger DNA.
5. Changes in the electrophoresis system will affect DNA migration, and it is necessary to add DNA standard reference materials for judgment.
6. The salt concentration in DNA samples will affect the mobility of DNA. Parallel control samples should use the same buffer conditions to eliminate this effect.
7. DNA mobility depends on the concentration of agarose gel, the shape and size of migrating molecules. It is possible to resolve a wide range of DNA molecules using gels of different concentrations, and agarose gels can be prepared to determine the concentration of the gel based on the range of DNA molecules. Polyacrylamide gel electrophoresis should be used for small fragment DNA electrophoresis to improve resolution.