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

Shipping and Storage

-Store at -30~-15 ℃. Transportation condition: ≤ 0 ℃.

Splice site

5'-GC↓GGCCGC-3'
3'-CGCCGG↑CG-5'

Quality Control

1. 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-)。
2. 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.
3. 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.
4. 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

• 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

1. It is sensitive to mammalian genomic DNA CpG methylation.
2. Star activity may appear after digestion for more than 16 hours
3. The amount of Not I enzyme required for complete digestion of the supercoiled plasmid is 5 times that of linear DNA