How does one perform a double or multiple restriction enzyme digestion?

If all restriction enzymes in a double or multiple digestion have the same optimal buffer, performing the digestion is straightforward. Simply add the enzymes such that the sum of the enzyme additions does not exceed 10% of the reaction volume.

For example:

*The Acetylated BSA supplied with Promega’s restriction enzymes is 10mg/ml, and therefore, should be diluted 1:10 with Nuclease-Free Water prior to use. Alternatively, the 10mg/ml BSA may be diluted 1:100 directly into the restriction enzyme digestion.

However, when two or more enzymes do not share an optimal buffer, several options are available:

1. Consult the restriction enzyme buffer compatibility tables as indicated above. Compare the activity of each enzyme in the optimal buffer of the other one. In many cases, good activity can also be obtained using one of Promega’s 4-CORE^® 10X Buffers or MULTI-CORE™ Buffer. If one of the enzymes exhibits <75% activity in a given buffer, the reaction time or units of enzyme may be increased. However, be aware of possible star activity.
2. Choose an isoschizomer or neoschizomer with more compatible buffer requirements. Please see Table 6 (Isoschizomers and Neoschizomers of Commercially Available Restriction Enzymes) of the Restriction Enzyme Properties.
3. Perform the first restriction digestion using the enzyme with the lower salt requirements. Following incubation, heat inactivate this enzyme (65°C for 10 minutes). Note that some enzymes are thermal stable. Then add the appropriate volume of water, 10X reaction buffer for the second enzyme, BSA (1mg/ml) and second enzyme such that the first reaction is diluted 1:5. Incubate at the appropriate temperature for the second restriction enzyme.
4. Perform each digestion sequentially using the optimal buffers. Following the first digestion, purify the plasmid DNA using the Wizard^® DNA Clean-Up System^(a) (Cat.# A7280) or heat inactivate, ethanol precipitate and then perform the second digestion.

^(a)U.S. Pat. No. 5,658,548 and other patents.

4-CORE and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. MULTI-CORE is a trademark of Promega Corporation.