DNA Polymerase T4, Exo-Minus
|Cloning||Storage: -20°CShipping: Blue Ice|
Catalyzes 5’=>3’ synthesis of DNA. Enzyme has 3’=>5’ exonuclease activity but lacks 5’=>3’ exonuclease activity. T4 DNA polymerase possessing 3'=>5' exonuclease activity exhibits greater activity on single-stranded DNA than on double-stranded DNA.
E. coli with a cloned gene43 of bacteriophage T4.
• Blunting of DNA ends: fill-in 5'-overhangs or/and removal of 3'-overhangs, see protocol
• Blunting of PCR products with 3’-dA overhangs.
• Synthesis of labeled DNA probes by the replacement reaction.
• Oligonucleotide-directed site-specific mutagenesis.
• Ligation-independent cloning of PCR products.
5X Reaction Buffer:
D3943-05: DNA Polymerase T4 Buffer, 5X: Supplied as a liquid in 335mM Tris-HCl, pH 8.8 at 25°C, 33mM magnesium chloride, 5mM DTT, 84mM (NH4)2SO4.
One unit of T4 DNA Polymerase catalyzes the incorporation of 10nmoles of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30min at 37°C.
Assayed in the following mixture: 67mM Tris-HCl, pH 8.8, 6.7mM MgCl2, 1mM DTT, 16.7mM (NH4)2SO4, 0.2mg/ml BSA, 0.033mM of each dNTP, 0.4meq/ml [3H]-dTTP, 0.2mM heat-denatured and nuclease-digested calf thymus DNA.
No conversion of covalently closed circular DNA to nicked DNA was detected after incubation of 10 units of T4 DNA Polymerase with 1ug of pUC19 DNA in 50ul reaction buffer for 4 hours at 37°C.
Storage and Stability: Aliquot to avoid repeated freezing and thawing.. Store at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Form: Supplied as a liquid in 20mM potassium phosphate, pH 7.5, 200mM potassium chloride, 2mM DTT, 50% glycerol.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
1.Sambrook, J., Fritch, E.F., Maniatis, T., Molecular Cloning: A Laboratory Manual, the second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, 5.44-5.47, 1989. 2. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 3.5.11-3.5.12, 1994-1997. 3. Challberg, M.D., Englund, P.T., Meth. Enzymol., 65, 39-43, 1980. 4. Kunkel, I.A., Roberts, J.D. and Zakour, R.A., Methods Enzymol., 154, 367-382, 1987. 5. Wang, et al., BioTechniques, 17, 236, 1994.