Manual

Product Number: NC01

Shipping and Storage

Stored at -20ºC, valid for two years.

Components

Component	NC01 30μl
NcoI	30μl
10×Cut Buffer	1ml
Easy-Load 10×Cut Buffer	1ml

Description

NcoI is a high-quality restriction endonuclease that has been genetically engineered and can quickly complete DNA cleavage using only one buffer within 5-15 minutes.Suitable for rapid enzymatic digestion of plasmid DNA, PCR products, or genomic DNA.

1. Enzyme activity detection: At the optimal reaction temperature, in a 20μl reaction system, 1μl NcoI can completely digest 1μg of plasmid containing a single NcoI cleavage site within 15 minutes.
2. Long term enzyme digestion detection: Incubate 1μl NcoI with 1μg λDNA for 3 hours at the optimal reaction temperature, and no non-specific degradation of the substrate caused by other nucleases contamination or star activity was detected. Delayed enzyme digestion may result in star activity.
3. Enzyme digestion ligation re digestion detection: At the optimal reaction temperature, use 1μl NcoI to digest the substrate, recover the enzyme digestion product, and use an appropriate amount of T4 DNA Ligase at 22℃ to reconnect the enzyme digestion product. After recovering the ligation product again, use the same endonuclease to cleave the ligation product again.
4. Detection of non-specific endonuclease activity: at the optimal reaction temperature, 1μl NcoI and 1μg super spiral plasmid DNA were incubated together for 4h, and agarose gel electrophoresis was used to detect that the plasmid DNA was still in the super spiral state.
5. Blue and white spot detection: The vector containing a single lacZ α gene was digested with 1μl NcoI, reconnected, and transformed into competent E. coli cells, and coated on LB medium plates containing corresponding antibiotics, IPTG, and X-gal. Products with correct connections will grow blue colonies, while products with incorrect connections (i.e. incomplete DNA end incisions) will grow white colonies. For NcoI, the proportion of white colonies should be less than 1%.

Figure 1. Experimental results of NcoI enzyme activity.The 20μl reaction system contains 1μg of pTac His MBP PreScission (prokaryotic active protein high expression plasmid) (6669bp), as well as 1μl of NcoI without or with the addition of 1X Cut Buffer and 1X Easy Load Cut Buffer, respectively. Incubate at 37°C for 15 minutes for enzyme cleavage reaction, incubate at 80°C for 20 minutes to inactivate the enzyme, then electrophoresis and use NA Red (EB upgraded product), Perform nucleic acid staining at 2000X and then perform fluorescence imaging analysis. The DNA marker used is DNA Ladder (0.2-12kb, 12 bands). The actual detection effect may vary due to differences in experimental conditions, detection instruments, etc. The effect shown in the figure is for reference only.

Basic information

Recognition sequence	Isoschizomer	Enzyme digestion temperature	Deactivation conditions	Methylation interference?
5'-C^CATGG-3' 3'-GGTAC^C-5'	Bsp19I	37℃	80ºC 20min	None

The activity (buffer compatibility) in different reaction buffers is as follows:

10×Cut Buffer	Easy-Load 10×Cut Buffer	Thermo FastDigest Buffer	NEB CutSmart® Buffer	Takara QuickCut™ Buffer
100%	100%	100%	100%	100%

Please refer to the table below for the methylation effects of NcoI recognition sites:

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

Features

1. Enzymatic cleavage can be completed within 5-15 minutes;
2. All endonucleases share a single enzyme digestion buffer, Cut Buffer, greatly simplifying the enzyme digestion reaction system and facilitating double or multiple enzyme digestion;
3. In response to the issue of differences in activity of different enzymes in Cut Buffer, the concentrations of different enzymes were adjusted to uniformly add 1μl of enzyme per 20μl of system for enzyme digestion reaction;
4. Many modifying enzymes, such as Alkaline Phosphatase, Antarctic Phosphatase, T4 DNA Ligase, T4 Polynucleotide Kinase, T4 PNK (3 'phosphatase minus), etc., are 100% compatible with Cut Buffer, making reaction systems such as "enzyme cut connect" and "enzyme cut modify connect" compatible and supporting single tube reactions;
5. Good enzyme activity redundancy makes it easy to cope with substrate excess or difficult template enzyme digestion.