Cat.No.：D23091A
Price: ￥
Specifications：
Zymagen RapidCut™系列限制性内切酶，15~30分钟快速酶切，通用缓冲液简化酶切体系，高酶活冗余应对复杂模板，适用于质粒、PCR产物及基因组DNA的快速酶切。
Fast enzymatic cleavage capability: The enzymatic cleavage reaction can be completed within 15 to 30 minutes, significantly shortening the experimental process and improving the efficiency of molecular cloning;
Universal buffer system: All endonucleases in the series share the same cleavage buffer "Buffer", simplifying the configuration of the reaction system, facilitating the design of double or multiple enzyme cleavage combinations, and eliminating the need for additional adjustment of buffer conditions;
Uniform enzyme addition standard: Considering the activity differences of various enzymes in the Buffer, the concentrations of each enzyme have been optimized and adjusted. All reactions can be carried out according to the uniform standard of adding 1 μL of enzyme for every 20 μL of the system, simplifying the operation steps and reducing calculation errors;
High enzyme activity redundancy: There is sufficient enzyme activity redundancy. Even in complex enzymatic cleavage scenarios such as excessive substrates or difficult templates, it can still maintain stable and efficient enzymatic cleavage performance.
| Cat. No. | Component | Specification | Quantity |
| B38011A | NcoⅠ | 500 U | 1 |
| B38021A | RapidCut™ Buffer (10×) | 1 mL | 1 |
Molecular experiments including vector construction, linearization of plasmid templates, site-directed mutagenesis screening, and assembly of long fragment genomic DNA.
The substrate DNA was not cut by the enzyme used. Why is that?
The following situations can be considered: (1) There are no recognition and cleavage 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. (2) The A or C at the recognition site of the restriction enzyme is methylated. Some restriction enzymes are sensitive to whether the base at the recognition site is methylated, and thus cannot cut this site. For the influence of methylation on the restriction enzyme's cleavage reaction, please refer to the table about the effect of methylation on the activity of restriction enzymes, which provides detailed explanations. (3) The substrate DNA is not pure. If the substrate DNA contains restriction enzyme inhibiting substances, it will affect the cleavage action of the restriction enzyme. In this case, the substrate DNA must be re-purified. (4) The position of the recognition and cleavage sites of the restriction enzyme in the advanced structure of the substrate DNA also has a certain impact on the cleavage reaction. (5) If there are no protective bases before the restriction enzyme cleavage site of the PCR primers or if the primer synthesis is incorrect, there will be no correct cleavage site. Before enzymatic cleavage of the PCR product, try to refine it to replace the Buffer to avoid other substances introduced by PCR products affecting the cleavage reaction. It has been reported that usually, the addition amount of PCR products to the total reaction volume below 25% is not a problem. (6) Do not cross-use enzymes and Buffers from different companies. Otherwise, it may affect the cleavage effect.
Can restriction enzymes be diluted for use?
It's better not to dilute. Restriction enzymes have been prepared with specific concentrations according to the optimal reaction system. When diluting the enzyme product, there is no linear relationship between enzyme concentration and activity. The lower the enzyme concentration, the lower the relative activity, and it becomes more unstable. Sometimes, the substrate DNA may not be cleaved.
The definition of 1 U of restriction enzyme activity is: at 37℃ for 1 hour, the amount of enzyme that completely cuts 1 μg of λ DNA, can it completely cut all the substrate DNA?
Due to the differences in the substrates' DNA, the molar concentrations of the enzyme cleavage sites are different, and the spatial structures are also distinct. All these factors will affect the speed of the enzymatic cleavage reaction. Therefore, the same amount of enzyme may not be able to cleave different substrates.
After the substrate DNA is subjected to enzymatic digestion, there are no bands or a smear phenomenon observed in the electrophoresis. How to solve this problem?
If DNase is present in the substrate DNA or the enzyme digestion reagent, under the influence of a certain temperature or buffer solution, the DNase will be activated and degrade the substrate DNA. Other restriction enzymes can be used to digest the substrate DNA, or this enzyme can be used to digest other substrate DNA to verify whether there is a problem with the enzyme and the substrate DNA. If this situation occurs during plasmid digestion, first perform ethanol precipitation on the plasmid before enzymatic digestion.
Can the enzyme-cut products be extracted with phenol/chloroform, recovered by gel cutting and using the fragment recovery kit?
The above purification methods can all be used to purify the enzyme-cut products. However, the losses in the phenol/chloroform extraction and gel cutting recovery are relatively large. The intermediate products of stepwise enzyme cleavage can be purified using ethanol precipitation and fragment recovery kits (using within the allowed length range for purification). This operation can reduce the losses.
Can the reaction system of restriction enzymes be made smaller?
It is not recommended to reduce the size of the enzyme digestion reaction system. A too-small enzyme digestion system may affect the enzymatic digestion outcome.
Can restriction enzymes be used for long-term enzymatic digestion reactions?
The enzyme digestion time should not be too long. If you need to extend the enzyme digestion time, it is recommended that the duration should not exceed "the time when no Star Activity occurs". Specific information can be found in the product manual.
What is the amount of restriction enzyme to be used during the double enzyme digestion? If the reaction temperatures for the two enzymes are different, how should it be operated?
When using restriction enzymes for double digestion, the total amount of the two enzymes should not exceed 1/10 of the reaction system. If the reaction temperatures of each enzyme are different, it is recommended to add the corresponding QuickCut restriction enzymes in the order from low temperature to high temperature for a stepwise enzymatic digestion reaction.
What is the optimal amount, concentration and duration of time for using restriction enzymes and DNA substrates?
The standard usage amounts of each restriction enzyme and DNA substrate should be referred to in the "General Reaction System" section of 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, it should be controlled 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.
