Universal buffers for double digestion with restriction enzymes
Double digestion (digesting DNA with two restriction enzymes simultaneously) is frequently performed to save time. Our restriction enzymes include universal buffers (refer to the restriction enzyme buffer activity page for relative activity in each buffer), but for some double digests, it may be difficult to select a buffer that is suitable for both enzymes. Buffer selection is critical for obtaining high activity of both enzymes and for avoiding star activity.
The table below lists the appropriate buffer, dilution factor, and necessary additive for common double digestions. Note, L, M, H, T and K indicate the type of buffer. The recommended final buffer concentration is also indicated (universal buffers are supplied at 10X concentration). BSA is supplied at 10X concentration; for use, dilute 10-fold to obtain a final concentration of 0.01%.
| Enzyme | Acc I | BamH I | Bgl II | Cla I | EcoR I | EcoR V | Hinc II | Hind III | Kpn I | Nco I | Nde I |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Supplied Buffer |
10X M | 10X K | 10X H | 10X M | 10X H | 10X H | 10X M | 10X M | 10X L | 10X K + BSA |
10X H |
| Acc I | – | 0.5X K | 1X T | 1X M | 1X M | 0.5X K | 1X M | 1X M | 1X M | 1X M + BSA |
1X T |
| BamH I | 0.5 × K | – | 1X K | 1X K | 1X K | 1X K | 0.5X K | 1X K | 0.5X K | 1X K + BSA |
1X K |
| Bgl II | 1X T | 1X K | – | 1X H | 1X H | 1X H | 2X K | 1X K | 1X T | 1X K + BSA |
1X H |
| Cla I | 1X M | 1X K | 1X H | – | 1X H | 1X H | 1X M | 1X M | 1X M | 1X K + BSA |
1X H |
| EcoR I | 1X M | 1X K | 1X H | 1X H | – | 1X H | 1X M | 1X M | 1X M | 1X K + BSA |
1X H |
| EcoR V | 0.5X K | 1X K | 1X H | 1X H | 1X H | – | 2X T | 1X K | 0.5X K | 1X K + BSA |
1X H |
| Hinc II | 1X M | 0.5X K | 2X K | 1X M | 1X M | 2X T | – | 1X M | 1X M | 1X M + BSA |
1X T |
| Hind III | 1X M | 1X K | 1X K | 1X M | 1X M | 1X K | 1X M | – | 1X M | 1X K + BSA |
1X K |
| Kpn I | 1X M | 0.5X K | 1X T | 1X M | 1X M | 0.5X K | 1X M | 1X M | – | 0.5X K + BSA |
1X T |
| Nco I | 1X M + BSA |
1X K + BSA |
1X K + BSA |
1X K + BSA |
1X K + BSA |
1X K + BSA |
1X M + BSA |
1X K + BSA |
0.5X K + BSA |
– | 1X K + BSA |
| Nde I | 1X T | 1X K | 1X H | 1X H | 1X H | 1X H | 1X T | 1X K | 1X T | 1X K + BSA |
– |
| Not I | 0.5X K + BSA |
0.5X K + BSA |
1X H + BSA |
1X H + BSA |
1X H + BSA |
1X H + BSA |
0.5X K + BSA |
0.5X K + BSA |
0.5X K + BSA |
0.5X K + BSA |
1X H + BSA |
| Pst I | 1X M | 1X K | 1X H | 1X H | 1X H | 1X H | 1X M | 1X M | 1X M | 1X K + BSA |
1X H |
| Pvu I | 0.5X K | 1X K | 1X K | 1X K | 1X K | 1X K | 0.5X K | 1X K | 0.5X K | 1X K + BSA |
1X K |
| Sac I | 1X M | 0.5X K | 0.5X K | 1X M | 1X M | 0.5X K | 1X M | 1X M | 1X L | 0.5X K + BSA |
1X T |
| Sal I | 1.5X T | 1.5X T | 1X H | 1X H | 1X H | 1X H | 1.5X K | 1.5X K | 1.5X T + BSA |
1.5X T + BSA |
1X H |
| Sma I | 1X T + BSA |
0.5X T + BSA |
1X T + BSA |
1X T + BSA |
1X T + BSA |
0.5X K + BSA |
1X T + BSA |
1X T + BSA |
1X T + BSA |
1X T + BSA |
1X T + BSA |
| Spe I | 1X M | 1X K | 1X H | 1X M | 1X H | 1X H | 1X M | 1X M | 1X M | 1X K + BSA |
1X H |
| Sph I | 0.5X K | 1X K | 1X H | 1X H | 1X H | 1X H | 2X T | 1X K | 0.5X K | 1X K + BSA |
1X H |
| Xba I | 1X M | 0.5X K | 2X T | 1X M | 1X M | 2X T | 1X M | 1X M | 1X M | 1X M + BSA |
1X T |
| Xho I | 1X M | 1X K | 1X H | 1X H | 1X H | 1X H | 1X M | 1X M | 1X M | 1X K + BSA |
1X H |
| Enzyme | Not I | Pst I | Pvu I | Sac I | Sal I | Sma I | Spe I | Sph I | Xba I | Xho I |
|---|---|---|---|---|---|---|---|---|---|---|
| Supplied Buffer |
10X H + BSA + Triton |
1X H | 10×K + BSA |
10X L | 10X H | 10X T + BSA |
10X M | 10X H | 10X M + BSA |
10X H |
| Acc I | 0.5X K + BSA |
1X M | 0.5X K | 1X M | 1.5X T | 1X T + BSA |
1X M | 0.5X K | 1X M | 1X M |
| BamH I | 0.5X K + BSA |
1X K | 1X K | 0.5X K | 1.5X T | 0.5X T + BSA |
1X K | 1X K | 0.5X K | 1X K |
| Bgl II | 1X H + BSA |
1X H | 1X K | 0.5X K | 1X H | 1X T + BSA |
1X H | 1X H | 2X T | 1X H |
| Cla I | 1X H +BSA |
1X H | 1X K | 1X M | 1X H | 1X T +BSA |
1X M | 1X H | 1X H | 1X H |
| EcoR I | 1X H + BSA |
1X H | 1X K | 1X M | 1X H | 1X T + BSA |
1X H | 1X H | 1X M | 1X H |
| EcoR V | 1X H + BSA |
1X H | 1X K | 0.5X K | 1X H | 0.5X K + BSA |
1X H | 1X H | 2X T | 1X H |
| Hinc II | 0.5X K + BSA |
1X M | 0.5X K | 1X M | 1.5X K | 1X T + BSA |
1X M | 2X T | 1X M | 1X M |
| Hind III | 0.5X K + BSA |
1X M | 1X K | 1X M | 1.5X K | 1X T + BSA |
1X M | 1X K | 1X M | 1X M |
| Kpn I | 0.5X K +BSA |
1X M | 0.5X K | 1X L | 1.5X T +BSA |
1X T +BSA |
1X M | 0.5X K | 1X M | 1X M |
| Nco I | 0.5X K + BSA |
1X K + BSA |
1X K + BSA |
0.5X K + BSA |
1.5X T + BSA |
1X T + BSA |
1X K + BSA |
1X K + BSA |
1X M + BSA |
1X K + BSA |
| Nde I | 1X H + BSA |
1X H | 1X K | 1X T | 1X T + BSA |
1X H | 1X H | 1X T | 1X H | 1X H |
| Not I | – | 1X H + BSA |
2X K + BSA |
0.5X K + BSA |
1X H + BSA |
0.5X T + BSA |
1X H + BSA |
1X H + BSA |
0.5X K + BSA |
1X H + BSA |
| Pst I | 1X H + BSA |
– | 1X K | 1X M | 1X H | 0.5X T + BSA |
1X H | 1X H | 1X M | 1X H |
| Pvu I | 2X K + BSA |
1X K | – | 0.5X K | 1.5X K + BSA |
1X K + BSA |
1X K | 1X K | 0.5X K | 1X K |
| Sac I | 0.5X K + BSA |
1X M | 0.5X K | – | 1.5X T + BSA |
1X T + BSA |
1X M | 0.5X K | 1X M | 1X M |
| Sal I | 1X H + BSA |
1X H | 1.5X K + BSA |
1.5X T + BSA |
– | 1.5X T + BSA |
1X H | 1X H | 1.5X T | 1X H |
| Sma I | 0.5X T + BSA |
0.5X T + BSA |
1X K + BSA |
1X T + BSA |
1.5X T + BSA |
– | 1X T + BSA |
0.5X T + BSA |
1X T + BSA |
1X T + BSA |
| Spe I | 1X H + BSA |
1X H | 1X K | 1X M | 1X H | 1X T + BSA |
– | 1X H | 1X M | 1X H |
| Sph I | 1X H + BSA |
1X H | 1X K | 0.5X K | 1X H | 0.5X T + BSA |
1X H | – | 2X T | 1X H |
| Xba I | 0.5X K + BSA |
1X M | 0.5X K | 1X M | 1.5X T | 1X T + BSA |
1X M | 2X T | – | 1X M |
| Xho I | 1X H + BSA |
1X H | 1X K | 1X M | 1X H | 1X T + BSA |
1X H | 1X H | 1X M | – |
Notes:
- Ten units of each enzyme completely digests 1 µg of DNA at 37°C in one hour in 50 µl reaction mixture.
- The concentration of glycerol should be less than 10% to minimize star activity.
- DNA may not be digested completely when recognition sequences of two enzymes are close each other for DNAs with high-structure conformations.
Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2025 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.
