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Activity of DNA/RNA Modifying Enzymes in Buffers for Restriction Enzymes, PCR, RT

Fermentas DNA/RNA modifying enzymes are supplied with optimized reaction buffers. However, it is often convenient to use these enzymes in other buffers for experiments that involve multiple enzymatic reactions.

The table below presents activities of DNA/RNA modifying enzymes in popular reaction buffers, supplied with Fermentas enzymes and used in common applications.

DNA/RNA modifying enzyme

Enzyme activity in 1X buffers, %
B G O R Tango™ 
1X		 Tango™
2X		 BamHI Ecl136II,
SacI		 EcoRI KpnI FastDigest® Taq buf.
with KCl		 Taq buf. with
(NH4)2SO4		 Pfu
buffer		 RT
buffer
DNA Polymerase I, E.coli 25-50 75-100 100 100 100 100 100 50-75 100 50-75 100 100 100 100 100
Klenow Fragment 25-50 25-50 100 100 100 100 100 50-75 100 50-75 100 100 100 100 100
Klenow Fragment, exo- 25-50 25-50 100 100 100 100 100 50-75 100 50-75 100 100 100 100 100
T4 DNA Polymerase 75-100 75-100 100 100 100 100 100 100 100 100 100 50 100 50 100
T7 DNA Polymerase 75-100 100 100 100 100 100 75-100 75-100 100 100 100 nd nd nd nd
Exonuclease I, E.coli nd nd nd nd nd nd nd nd nd nd nd 100 100 100 nd
Exonuclease III 100 25-50 0-25 0-25 25-50 0-25 0-25 100 0-25 100 0-25 nd nd nd nd
T4 DNA Ligase*  100 100 75-100 75-100 75-100 75-100 75-100 50 75-100 100 75-100 75 75 75 75
FastAP™ Thermosensitive Alkaline Phosphatase 100 100 100 100 100 100 100 100 100 100 100 100 50 75-100 100
Shrimp Alkaline Phosphatase (SAP) 100 100 100 100 100 100 100 75-100 100 100 100 100 100 100 100
Calf Intestine Alkaline Phosphatase (CIAP) 100 100 100 100 100 100 100 25-50 100 100 100 100 100 100 100
T4 Polynucleotide Kinase** 75-100 100 100 75-100 100 100 100 50-75 100 75-100 100 100 0 0 100

* Buffers were supplemented with 0.5 mM ATP, required for T4 DNA Ligase activity.
** The activity of this enzyme was compared to activity in buffer A (for forward reaction).
nd - Not determined.

1X Buffer B: 
 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 0.1 mg/ml BSA.

1X Buffer G: 
 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, 0.1 mg/ml BSA.

1X Buffer O: 
 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.1 mg/ml BSA.

1X Buffer R: 
 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA.

Buffer Tango™
1X: 33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate, 0.1 mg/ml BSA.
2X: 66 mM Tris-acetate (pH 7.9 at 37°C), 20 mM Mg-acetate, 132 mM K-acetate, 0.2 mg/ml BSA.

1X Buffer BamHI: 
 10 mM Tris-HCl (pH 8.0 at 37°C), 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100, 0.1 mg/ml BSA.

1X Buffer Ecl136II, SacI: 
 10 mM Bis-Tris Propane-HCl (pH 6.5 at 37°C), 10 mM MgCl2, 0.1 mg/ml BSA.

1X Buffer EcoRI: 
 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100, 0.1 mg/ml BSA.

1X Buffer KpnI: 
 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 0.02% Triton X-100, 0.1 mg/ml BSA.

1X Taq Buffer with KCl:
10 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl2, 50 mM KCl, 0.08% Nonidet P40.

1X Taq Buffer with (NH4)2SO4:
75 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgCl2, 20 mM (NH4)2SO4, 0.01% Tween 20.

1X Pfu Buffer:
20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1 mg/ml BSA.

1X RT Buffer:
50 mM Tris-HCl (pH 8.3 at 25°C), 4 mM MgCl2, 50 mM KCl, 10 mM DTT.

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Updated sausio 31, 2008 14:46