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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.

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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.

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