Takara
login | register | order now | view cart | feedback
Support
Applications


Support >  Applications >  Restriction_Enzymes >  Double_Digestion_Buffers

Recommended Universal Buffer for Double Digestion

Recommended Universal Buffer for Double Digestion

Double digestion, digesting DNA with two restriction enzymes simultaneously, is frequently performed to save time. Restriction enzymes from Takara Bio include universal buffers (refer to the Buffer Activity Chart 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. Use the table below to find the appropriate buffer, dilution factor, and necessary additive for common double digestions.

In the tables below, L, M, H, T and K indicates the buffer type. 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%.

See the second chart for Not I to Xho I.

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

Note:

  1. Ten units of each enzyme completely digests 1 µg of DNA at 37°C in one hour in 50 µL reaction mixture.
  2. The concentration of glycerol should be less than 10% to minimize star activity.
  3. DNA may not be digested completely when recognition sequences of two enzymes are close each other for DNAs with high-structure conformations.

^ to top

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

Note:

  1. 10 units of each enzyme completely digests 1 µg of DNA at 37°C in one hour in 50 µl reaction mixture.
  2. The concentration of glycerol should be less than 10% to minimize star activity.
  3. DNA may not be digested completely when recognition sequences of two enzymes are close each other for DNAs with high-structure conformations.

^ to top

Clontech is a Takara Bio Company © 2014 Privacy Policy | Terms & Conditions