T4 DNA Polymerase is a high-fidelity enzyme with robust 5′→3′ polymerase and strong 3′→5′ exonuclease activities. It is ideal for generating blunt-ended DNA, removing 3′ overhangs, filling in 5′ overhangs, DNA labeling, cloning, and site-directed mutagenesis.
Product Description
IG® T4 DNA Polymerase has both a DNA-dependent DNA polymerase activity and a potent 3´→5´ exonuclease activity.
The purity of this enzyme is >95% homogeneity as determined by SDS-PAGE using Coomassie® Blue staining (see figure below).
Product Source
E. coli cells with a cloned gene of bacteriophage T4 DNA Polymerase.
Applications
- 3´-overhang removal to form blunt ends (1, 2).
- 5´-overhang fill-in to form blunt ends (1, 2).
- Probe labeling using replacement synthesis (2).
- DNA library preparation for Next-generation sequencing.
- Ligation-independent cloning of PCR products.
- Second strand synthesis in site-directed mutagenesis (3).
Product Includes
- T4 DNA Polymerase
- 10x T4 DNA Polymerase Buffer
Storage Temperature
–20 °C
Storage Buffer
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA,
50% Glycerol, pH 7.5 @ 25 ºC
10X T4 DNA Polymerase Reaction Buffer
500 mM Tris-HCl, 100 mM MgCl2, 50 mM dithiothreitol, pH 7.5 @ 25°C.
Unit Definition
One unit of T4 DNA Polymerase converts 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 37°C under standard assay conditions.
Inactivation
Inactivated by heating at 70°C for 15 min.
Quality Control Assays
IG™ T4 DNA Polymerase is free from detectable endonuclease and RNase activities.
Technical Support
Intact Genomics is committed to supporting the worldwide scientific research community by supplying the highest quality reagents. Each new lot of our products is tested to ensure they meet the quality standards and specifications designated for the product. Please follow the instructions carefully and contact us if additional assistance is needed. We appreciate your business and your feedback regarding the performance of our products in your applications.
Blunting ends by 3’ overhang removal or 3’ recessed end fill-in:
1. Dissolve DNA in 1x reaction buffer supplemented with 100 μM dNTPs.
2. Add 1 unit T4 DNA Polymerase per μg DNA.
3. Incubate at room temperature for 5-30 minutes. Stop reaction by heating at 70°C for 20 minutes.
References
1. Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc.
2. Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
3. Kunkel, T.A. et al. (1987). Methods Enzymol. 154, 367-382.
Manual – T4 DNA Polymerase
MSDS – IG T4 DNA Polymerase