Experimental Techniques

The success rate will be affected by the following factors:

The distance between the mutation sites (sites that are farther apart have a higher efficiency); 

The type of mutation (base substitution is more efficient than insertion/deletion); 

The size of the plasmid and the complexity of the sequence; 

It is recommended to conduct a small-scale preliminary experiment before the formal experiment to evaluate the efficiency.

The following situations can be considered:

• If there are no recognition and cutting sites for the restriction enzyme on the substrate DNA. Especially for some DNA that has undergone recombination and other treatments, the bases are prone to deletion and change.

• The A or C at the recognition site of the restriction enzyme is methylated. Some restriction enzymes are sensitive to whether the bases at the recognition site are methylated, and thus cannot cut at that site. For the influence of methylation on the restriction enzyme's cutting reaction, please refer to the table showing the effect of methylation on the activity of restriction enzymes, which provides detailed explanations.

• The substrate DNA is not pure. If the substrate DNA contains restriction enzyme inhibiting substances, it will affect the cutting action of the restriction enzyme. In this case, the substrate DNA must be re-purified.

• The position of the recognition and cutting sites of the restriction enzyme in the advanced structure of the substrate DNA also has an impact on the enzyme cutting reaction.

• The restriction enzyme recognition and cutting sites are located in the advanced structure of the substrate DNA. Their positions also have an influence on the enzyme cutting reaction.

• The restriction enzyme recognition and cutting sites of the PCR primers do not have protected bases or the primer synthesis is incorrect, resulting in no correct cutting sites. Before enzymatic cutting of the PCR product, try to refine it to replace the Buffer to avoid other substances introduced by PCR products affecting the enzyme cutting reaction. It has been reported that generally, the addition amount of PCR products in the total reaction volume below 25% is not a problem.

• Do not cross-use enzymes and Buffers of different brands. Otherwise, it may affect the enzyme cutting effect.

For the standard usage amounts of each restriction enzyme and DNA substrate, please refer to the "General Reaction System" section in the manual.

The activity unit of restriction enzymes is defined based on a 1-hour reaction. Generally, the recommended reaction time ranges from 1 hour to several hours. The standard enzyme volume used in the reaction is 1/20 of the reaction volume. If you want to increase the enzyme usage, please keep it below 1/10 of the reaction volume. If too much glycerol (more than 5%) is introduced into the reaction solution from the restriction enzyme solution, it will affect the recognition of the DNA sequence by the restriction enzyme, resulting in incorrect sequence recognition and non-specific enzyme digestion. If the enzyme amount (U) used is insufficient relative to the DNA substrate, a longer enzyme digestion reaction time is required. Additionally, the activity of restriction enzymes is affected by the sequences before and after the recognition site, and the difficulty of enzyme digestion varies.

EDTA can be added to the reaction system to a final concentration of ≥ 11 mM to chelate the Mg²⁺ ions in the reaction system, or DNA loading buffer containing SDS can be added to achieve a final concentration of SDS of 0.08%.