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  5. Thermo Scientific™ NcoI (10 U/μL)

Thermo Scientific™ NcoI (10 U/μL)

Product Code. 10233160 Shop All Thermo Scientific Products
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Product Code. 10233160 Supplier Thermo Scientific™ Supplier No. ER0571

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Description

The NcoI restriction enzyme recognizes C^CATGG sites and cuts best at 37°C in Tango buffer (Isoschizomers: Bsp19I).

Conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.

5'...C▵C A T G G...3'

3'...G G T A C▵C...5'

Conditions for 100% Activity:

  • 1X Buffer Tango: 33mM Tris-acetate (pH 7.9 at 37°C), 10mM Mg-acetate, 66mM K-acetate and 0.1mg/mL BSA
  • Incubate at 37°C

Storage Buffer:

  • NcoI is supplied in: 10mM Tris-HCl (pH 7.5 at 25°C), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/mL BSA and 50% (v/v) glycerol

Ligation and Recleavage:

  • After 50-fold overdigestion with NcoI, more than 95% of the DNA fragments can be ligated and recut

Methylation Effects:

  • Dam: never overlaps —no effect
  • Dcm: never overlaps —no effect
  • CpG: never overlaps —no effect
  • EcoKI: never overlaps —no effect
  • EcoBI: never overlaps —no effect

Digestion of Agarose-embedded DNA:

  • Minimum 5units of the enzyme are required for complete digestion of 1μg of agarose-embedded lambda DNA in 16 hours

Compatible Ends:

  • AflIII, PscI, BtgI, Eco130I, FatI, PagI

Note:

Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.

TRUSTED_SUSTAINABILITY

Specifications

Concentration 10 U/μL
Quantity 500 U
Product Type NcoI

Frequently Asked Questions (FAQs)

Can I double digest my DNA using a Thermo Scientific conventional restriction enzyme and a FastDigest restriction enzyme?

For optimal results with fast reaction and 100% buffer compatibility, we highly recommend using FastDigest restriction enzymes in double digestion. In certain cases however, it may be possible to perform double digestion using a mix of Thermo Scientific conventional and Fastdigest restriction enzymes. For specific recommendations, please contact our technical service with detailed information about the enzymes and DNA template you plan to use.

Why do you recommend only 2 µL of 10X Reaction Buffer when digesting unpurified PCR product in a 30 µL reaction?

We recommend only 2 µl 10X Buffer in digestion of unpurified PCR products in 30 ul since salts and ions from the PCR reaction would be carried over to the digestion reaction.
What are key factors promoting star activity?

Star activty may be contributed by:

• Prolonged incubation
• High enzyme concentration
• High glycerol concentration (usually 5% or higher)
• Small reaction volume
Unexpected DNA bands were observed on agarose gel electrophoresis after restriction digestion. What may have caused this?

Unexpected cleavage patterns may be caused by the following reasons:

• Star activity of the restriction enzyme: Make sure to follow the reaction recommendations as specified in the protocol. Star activity may be improved by changing several key factors such as decreasing the reaction time, increasing the reaction volume, and decreasing the enzyme amount.

• Partial or incomplete cleavage (incomplete restriction reaction): Efficiency of the enzyme can be improved by adding more enzyme, prolonging the reaction time, or purifying DNA samples to remove inhibitory contaminants.

• Contamination with non-specific endonucleases: Non-specific endonucleases may be introduced to the DNA sample and/or the enzyme from improper handling, pipetting, etc.

•Improper reaction setup: Mix the digestion reaction thoroughly.

What are possible reasons for incomplete/failed restriction digestion?

The main reason for DNA cleavage reaction failure is the presence of contaminating inhibitors in the template DNA (for example: phenol, chloroform, detergents, ethanol, excess salts, EDTA, etc.). The best way to troubleshoot is to perform control reactions:

1) negative control (experimental DNA in the reaction buffer without the restriction enzyme) to access degradation of DNA by contaminants in the DNA template and/or reaction buffer
2) positive control reaction I (digestion of highly pure control DNA with the restriction enzyme) to access reaction conditions and enzyme activity
3) positive control reaction II (highly pure control DNA + experimental DNA + Restriction Enzyme) to access possible issues with the experimental DNA.

In addition, please check for sensitivity of the restriction enzymes to template DNA methylation.
What is the optimal DNA concentration in restriction enzyme digestion?

The optimal range of DNA concentration is 0.02-0.1 µg/µL in the restriction digestion mixture. We would recommend the volume of the DNA sample not to exceed 30% of the total reaction volume to avoid potential reaction inhibition. To resolve inhibition from impurities of the DNA solution, we recommend increasing the overall volume of the reaction while keeping the volume of the DNA solution the same.

Is there any other way to inactivate Thermo Scientific restriction enzymes (when thermal inactivation is not applicable) without using phenol/chloroform?

To remove the enzymes without using phenol/chloroform, we would recommend silica column based purification such as GeneJET Gel Extraction and DNA Cleanup Micro Kit (Cat. Nos. K0831/2) or GeneJET PCR Purification Kit (Cat. Nos. K0701/2).
What is the stability of Thermo Scientific restriction enzymes if they freeze-thaw during shipment?

Enzymes may freeze during shipment on dry ice. This does not affect their quality as all Thermo Scientific enzymes are tested 100% active after at least three freeze-thaw cycles. For 24-48 hour delivery, enzymes may be shipped on blue ice because their quality is not affected by short exposure to 4 degrees C.
Do Thermo Scientific restriction enzymes come with the buffer(s)?

Thermo Scientific restriction enzymes are shipped with their optimal digestion buffer(s). The buffers are available separately for purchase as well.

For Research Use Only. Not for use in diagnostic procedures.

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