Protocol of double digestion of DNA by restriction endonucleases.

In accordance with this protocol, restriction endonucleases for rapid hydrolysis (Turbo) are used for double digestion of DNA. To hydrolyze DNA with two restriction endonucleases simultaneously, we recommend using the universal reaction buffer “ROSE” (B021), or one of the buffers for restriction endonucleases, in which both enzymes exhibit at least 50% activity. Otherwise, it is better to carry out sequential hydrolysis of DNA by enzymes. Information about the activity of restriction endonucleases in the ROSE buffer and in other reaction buffers can be found in the Service section/Restriction activity). Reaction Original SibEnzyme (ROSE) buffer is a specially designed universal reaction buffer for restriction endonucleases. The majority of restriction endonucleases exhibit 50-100% activity in it.

Standard protocol of double digestion of DNA by Turbo restriction endonucleases.

Total Volume 20 ul:

• Optimal or Universal reaction buffer (x10) – 2 ul;
• DNA sample  – 0.2 – 1 ug;
• BSA (10 mg/ml)   – 0,2 μl (for some enzymes)
• Nuclease-free water – to 20 μl
• + 1 μl of Restriction Endonuclease 1 and 1 μl of Restriction Endonuclease 2

Incubate at optimal temperature for 0,5-1 hour.

BSA should be added to the reaction mixture if at least one of the enzymes requires its addition to achieve maximum activity.

Note:

• The final concentration of glycerol in any reaction should be less than 5% to avoid the star activity. This means that no more than a total of 2 µl of enzymes should be added to the standard reaction mixture of 20 µl.
• If the enzymes have different optimal reaction temperatures, it is recommended to perform the hydrolysis reactions sequentially (starting with the enzyme with the lower optimal temperature), with inactivation of the restriction enzyme after the first hydrolysis.