Technical Data
D3943
DNA Polymerase T4, Exo-Minus
100u
5x100u
5x100u
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Technical Data |
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D3943 |
DNA Polymerase T4, Exo-Minus |
100u 5x100u |
| Cloning | Storage: -20°CShipping: Blue Ice |
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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. Source: E. coli with a cloned gene43 of bacteriophage T4. Applications: • 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. Unit Definition: 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. Activity Assay: 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. Quality Control: 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. Molecular Weight: ~104kD |
Concentration: ~5u/ul 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. |
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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.
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