Several major factors affect enzymatic cutting.

factors affecting enzyme cutting:
1. The quality < of > dna
1) is the most important factor in determining the efficiency of enzymatic cutting. Usually in cloning, if the enzyme cutting conditions are repeatedly optimized, no complete enzymatic cutting can be achieved, the most likely cause is a problem with the quality of the proton DNA.
2) DNA quality monitoring usually has two methods: first, the OD260/OD280 ratio should be around 1.8 (1.7-1.9), otherwise it means that there is a large amount ofprotein or RNA contamination in the DNA sample. Secondly, < analysis of > asugar electrophoresis should be mainly based on ultra-helix bands. No more than three bands (ultra-helix DNA, linearized DNA, and ring DNA, respectively). Otherwise, the quality of the granulate DNA is not high and should be re-prepared.

. 2.active inactive
1) restrictive endoenzymes generally need to be preserved at low temperatures, and the damage to enzyme activity is obvious from repeated cooling processes. Therefore, in order to ensure that the restricted endoenzyme does not insesterit during the validity period, the daily preservation and use of the restrictive endoenzyme should be very careful.
2) It is recommended to purchase a freezer box with insulation function to save the restrictive endoenzyme (-20 degrees), and when using the restrictive endoenzyme, you should also use the freezer box with insulation function, as far as possible to prevent the temperature of the enzyme repeated large fluctuations.

. 3. The unit definition of restrictive endoenzyme
1) restrictive endoenzyme is usually defined as one unit of enzyme required to fully digest a ugDNA substrate at a suitable temperature.
2) There are several uncertainties in this unit definition: first, the substrate, where different enzyme units are defined as the selected substrate may be different (several commonly used substrate DNA include: Lambda DNA, AD2 DNA, and some proton DNA); Because full digestion is required in the unit definition, the number of enzyme cut points on the substrate directly affects the unit definition of the enzyme.
3) Therefore, when enzymatic cutting, the usual practice of digesting 1ugDNA with 1ulase (generally 10IU/ul) is unscientific, which also leads to the fact that in practice, many pre-tests are required to determine the most suitable enzyme cutting conditions.
4) Before, I recommended an online dual enzymatic cutting design software, double digestion designer, that can accurately calculate the amount of restrictive endoenzyme at the time of enzymatic cutting. In use, it can be noted that the dosage of the two enzymes used for double enzymatic cutting sometimes differs by nearly 20 times (EcoRI and NheI), and it is found that small fragments PCR products (100-500bp) require more than 10 times the amount of enzymes required for mass DNA enzymatic cutting.
5) The software is currently free to use, the username and password are test. (Responsible Editor: King Kunlun, Great Han)