Vaccinia Capping Enzyme
2022-09-09
Duplex specific nuclease
2022-10-13
Description
Not I is a fast restriction endonuclease that can accurately complete DNA cutting within 5-15 minutes through genetic engineering recombination technology. It is suitable for fast cutting of plasmid DNA, PCR products, genomic DNA, etc.
Components
| Component | NT01 |
| Not I | 2500U |
| 10× Reaction Buffer | 1ml |
Shipping and Storage
-Store at -30~-15 ℃. Transportation condition: ≤ 0 ℃.
Splice site
5′-GC↓GGCCGC-3′
3′-CGCCGG↑CG-5′
Quality Control
- Functional activity test: under the optimal reaction temperature, at 20 μ L reaction system, 1 μ L enzyme can be completely digested in 15 min μ g λ DNA (Dam-)。
- Ultra long incubation test: under the optimal reaction temperature, set the μ L enzyme and 1 μ g λ DNA (Dam -) was incubated for 3 h, and no non specific degradation of substrate caused by other nuclease contamination or asterisk activity was detected, but the asterisk activity may occur if the incubation time is prolonged.
- Enzymatic digestion – linking – re digestion detection: under the optimal reaction temperature, use 1 μ L enzyme digested the substrate and recovered the digestion product. The digestion product can be reconnected by using an appropriate amount of T4 DNA ligase at 22 ° C. After the connection product is recovered again, the connection product can be re cut by using the same endonuclease.
- Detection of non-specific endonuclease activity: under the optimal reaction temperature μ L enzyme and 1 μ After incubation for 4 h with g super helix plasmid DNA, the plasmid DNA was still in the super helix state by agarose gel electrophoresis.
Reaction conditions
10× Reaction Buffer，37℃。
Thermal deactivation: 65 ℃ for 20 minutes.
Protocol
- Prepare the system reaction liquid according to the following recommended sample adding sequence (operation on ice):
Component 50 μL Reaction system
| Component | 50 μL Reaction system |
| DNA | 1 μg |
| Not I | 20 U |
| 10× Reaction Buffer | 5 μl |
| ddH2O | up to 50 μl |
- Incubate at 37 ° C for 5-15 min.
- Incubation at 65 ° C for 20 min can inactivate the enzyme and stop the reaction (optional).
Note
- It is sensitive to mammalian genomic DNA CpG methylation.
- Star activity may appear after digestion for more than 16 hours
- The amount of Not I enzyme required for complete digestion of the supercoiled plasmid is 5 times that of linear DNA
