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.

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.

Enzyme finder, a new tool available on our website in the sidebar under "Technical Information", can be used to search for restriction enzymes by name, sequence, overhang or type. 

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.

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℃.

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.

Star activty may be contributed by:

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

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.

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℃.

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.