LightNing® AgeI

 

     LightNing® enzymes are a series of engineered restriction enzymes that are capable of fast DNA digestion. All LightNing® enzymes show superior activity in the universal CutOne® and CutOne® Color Buffer, and are able to digest DNA in 5~15 minutes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. LightNing® enzymes have passed multiple strict quality controls, and can be used to digest plasmid, genomic and viral DNA as well as PCR products.

    CutOne® Color Buffer includes a density reagent along with red and yellow tracking dyes that allow for direct loading of the reaction mixtures on a gel. The red dye of the CutOne® Color Buffer migrates with 2.5 kb double-strand DNA fragments in a 1% agarose gel, and the yellow dye migrates with 10 bp double-strand DNA fragments in a 1% agarose gel.

 

 

The reaction can be completed within 15 minutes

Recommended Reaction Conditions
1× CutOne® Buffer；
Incubate at 37℃；
Refer to "Protocol for Fast DNA Digestion" for reaction setup.

Heat Inactivation
Incubation at 80℃ for 20 minutes.

Methylation sensitivity
Cleavage with this restriction enzyme may be blocked or impaired when the substrate DNA is methylated by the CpG methylase.

 

 

 

Q：How stable is a particular restriction enzyme?

 

A：After plenty of experiment with restriction enzymes, we have found that most are very stable when stored at -20°C in the recommended storage buffer. Exposure to temperatures above -20°C should be minimized whenever possible.

Q：How should I stop my restriction digest?

 

A：If no further manipulations of the digested DNA are planned, the reaction can be terminated by adding a stop solution. At Yugong，we use the following stop solution: 500 mM EDTA (pH 8.0)(5 μl / 50 μl reaction). If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 80°C for 20 minutes) is the simplest method of stopping a reaction. Since this method does not work for all restriction enzymes, refer to the catalog information for the particular enzyme(s) you are using. Phenol/chloroform extraction is another means of inactivating a restriction enzyme.

Q：Do degenerate recognition sites need to be palindromic?

 

A：Most restriction enzyme recognition sites are palindromic and include only specified base pairs (i.e., EcoRI recognizes GAATTC). However, some enzymes have degenerate sites, meaning that they contain one or more base pairs that are not specifically defined (i.e., AccI recognizes GTMKAC, where M= A or C and K= Gor T). For degenerate enzymes, any base represented by the single letter code may be present at either location in the recognition site for cleavage to occur. For example, AccI recognizes all of the following sequences: GTATAC, GTAGAC, GTCGAC, GTCTAC.

Q：What is the stability of LightNing enzymes if they freeze-thaw during shipment?

 

A：Enzymes may freeze during shipment on dry ice. This does not affect their quality as all LightNing 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℃.

Q：Unexpected DNA bands were observed on agarose gel electrophoresis after restriction digestion. What may have caused this?

 

A：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.

Q：What are key factors promoting star activity?

 

A：Star activty may be contributed by:

• Prolonged incubation
• High enzyme concentration
• High glycerol concentration (usually 5% or higher)
• Small reaction volume

Q：What are possible reasons for incomplete/failed restriction digestion?

 

A：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.

Q：Can I store the CutOne Buffers at 4℃?

 

A：The CutOne Buffers can be stored at 4℃ for at least 6 months. For long-term storage until their expiration, we recommend storing the CutOne and CutOne Color Buffers at -20℃.

Q：What is the difference between CutOne Buffer and CutOne Color Buffer?

 

A：The CutOne Color Buffer offers the same performance as the colorless CutOne Buffer but enables direct loading of the reaction mixture on gels. The 10× CutOne Color Buffer includes a density reagent and two tracking dyes for direct loading. The red dye migrates with 2.5 kb DNA fragments in a 1% agarose gel and the yellow dye migrates with 10 bp DNA fragments in a 1% agarose gel.

Q：How I can transform or calculate reaction numbers in the unit of LightNing enzymes?

 

A：The concentration of LightNing enzymes is proprietary information. One rxn LightNingt enzyme activity is defined as: 1 µl of enzyme cleaves 1 µg of DNA substrate in 15 min at optimum temperature(most are 37℃) in 20 µl of 1× CutOne buffer.

Q：Do LightNing restriction enzymes contain BSA?

 

A：No，we have replaced BSA with rHSA，which is a well defined, animal-free recombinant human albumin.

Q：How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

 

A：A minimum of 6 protected base pairs upstream and downstream of the cleavage site is generally required, with no specific sequence requirement.

Q：Do I have to set-up digests with LightNing® enzymes for 5-15 minutes? Can I digest longer?

 

A：LightNing® enzymes have the benefit of working fast (5-15 minutes), but are also designed and qualified to withstand longer（no more than 3h） digestions without degradation of DNA.