NdeⅠ-Biosci-SuperCut™ Dakewe

Restriction Enzyme Site

5’…C A↓T A T G…3′
3’…G T A T ↑A C…5′
Isoschizomers*: FauNDI
*Isoschizomers may have different methylation sensitivities.

 

Storage Condition

Storage at -25℃~-15℃ and validity for 24 months.
Shipped on Blue Ice.

 

Components

Components	Amount
SuperCut™ NdeⅠ (10 units/μL)	2000 units
SuperCut™ 10×Buffer	2×1 mL
6× Gel Loading Buffer	1 mL

 

 

Activity Definition

One unit is defined as the amount of enzyme required to digest 1 μg of λDNA in 50 μL the reaction at the optimal reaction temperature in 60 minutes.

 

Description

SuperCut™ Restriciton Enzymes are a series of genetically engineered restriction enzymes that can accurately complete DNA digestion in 5~15 minutes, suitable for rapid digestion of plasmid DNA, PCR products or genomic DNA.
SuperCut™ Restriciton Enzymes has the following characteris-tics: digestion can be completed within 5~15 minutes; share a diges-tion buffer, which greatly simplifies the digestion reaction system; good enzyme activity redundancy for easy substrate overload or com-plex template digestion. In addition, dephosphorylation and ligation reagent of Biosci TM is 100% active in SuperCut™ Buffer, supporting one-tube reaction and improving the experience of “digestion-ligation-redigestion”.

 

Recommended Reaction Conditions

1× SuperCut™ Buffer；
Incubate at 37℃；
Refer to “Protocol for Fast DNA Digestion” for reaction setup.

 

Heat Inactivation

1.Incubation at 80℃ for 20 minutes.
2.Add an appropriate amount of 6 × Gel Loading Buffer according to the reaction system to terminate the reaction.

 

Quality Control

Functional Test
A 20 μL reaction in SuperCut™ Buffer containing 1 μg of pUC19 DNA and 10 units of SuperCut™ NdeⅠ incubated for 15 minutes at 37℃ results in complete digestion as determined by agarose gel electrophoresis.

Prolonged Incubation / Star Activity Assay
A 20 μL reaction in SuperCut™ Buffer containing 1 μg of pUC19 DNA and 1 μL of SuperCut™ NdeⅠ incubated for 3 hours at 37℃ results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Longer incubation may result in star activity.

Enzyme Digestion-Ligation-Redigestion Test
At the optimal reaction temperature, the DNA was digested using 10 units of SuperCut™ NdeⅠ, and then the digestion product was recovered. The DNA fragments can be religated using an appropriate amount of T4 DNA Ligase at 22℃. After the ligation product is recovered again, the ligation product can be recut using SuperCut™ NdeⅠ.

Non-specific Endonuclease Activity
At the optimal reaction temperature, 10 units of SuperCut™ NdeⅠ was incubated with a 20 μL reaction in SuperCut™ Buffer containing 1 μg of supercoiled plasmid DNA for 4 h, and the plasmid DNA was still supercoiled detected using agarose gel electrophoresis.

Blue/White Screening Assay
An appropriate vector containing lacZα gene is digested by 10 units of SuperCut™ NdeⅠ. The digested product is ligated and transformed into E.coli competent cell. On Luria-Bertani culture plate with X-Gal, IPTG and appropriate antibiotic, the successfully ligated β-galactosidase gene can be expressed and gives rise to a blue colony, while an interrupted gene (i.e. degraded DNA end) gives rise to a white colony. SuperCut™ restriction enzymes must produce fewer than 1%
white colonies.

 

Method of application

1. Protocol for Fast DNA Digestion
① Combine the following reaction components on ice in the order indicated：

	Plasmid DNA	PCR product	Genomic DNA
DNA a	≤ 1 μg	≤0.2 μg	≤ 5 μg
SuperCut™  NdeⅠ	10 units	10 units	30-50 units
SuperCut™ 10×Buffer	2 μL	3 μL	5 μL
ddH₂O	Up to 20 μL	Up to 30 μL	Up to 50 μL

a. DNA should be free of phenol, chloroform, ethanol, EDTA, detergents or high concentrations of salts, otherwise enzyme activity will be affected; Methylated DNA inhibits certain restriction enzyme digestion reactions.

② Gently mix or flick the tube wall to mix well (never vortex), then centrifuge instantaneously to collect reaction solution；
③ Incubate at 37℃ for 15 minutes (plasmid DNA) or for 15~30 minutes (PCR product) or for 30~60 minutes (genomic DNA)；
④ Optional: Inactivate the enzyme by heating for 20 minutes at 80℃, and add an appropriate amount of 6× Gel Loading Buffer according to the reaction system to terminate the reaction.

2. Double and Multiple Digestion of DNA
① Use 10 units of each enzyme and scale up the reaction conditions appropriately；
② The combined volume of the enzymes in the reaction mixture should not exceed 1/10 of the total reaction volume；
③ If the enzymes require different reaction temperatures, start with the enzyme that requires a lower temperature, then add the second enzyme and incubate at the higher temperature.
Note: If the total volume of reaction solution is larger than 20 μL, the incubation time should be increased appropriately, and water bath, metal bath, or sand bath should be used as much as possible.

Number of Recognition Sites in DNA

λDNA	ΦX174	pBR322	pUC57	pUC18/19	SV40	M13mp18/19	Adeno2
7	0	1	1	1	2	3	2

 

Methylation Effects on Digestion

Dam	Dcm	CpG	EcoKI	EcoBI
No effect	No effect	No effect	No effect	No effect

 

Activity in Different Buffers

	SuperCut™ Buffer	Thermo Scientific FastDigest Buffer	NEB CutSmart ® Buffer	Takara QuickCut™ Buffer
Activity	100%	100%	100%	100%

Note: The activity data is from the standard reation test of Biosci™ Restriction Enzyme described above.