DNA Polymerase T4, a template-depended DNA polymerase, catalyzes 5=>3 synthesis from primed single stranded 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. The 35 exonuclease activity of T4 DNA Polymerase is stronger on single-stranded DNA than on double stranded DNA and greater (more than 200 times) than that of D3935 DNA Polymerase I, E.coli Source: E. coli with a cloned gene43 of bacteriophage T4. 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 37C. Supplied with: D3943-05: DNA Polymerase T4 Buffer, 5X: Supplied as a liquid in 335mM Tris-HCl, pH 8.8, 33mM magnesium chloride, 5mM DTT, 84mM (NH4)2SO4. 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. Inhibition and Inactivation: Inhibitors: metal chelators, nucleotide analogs 2(p-n-butylanilino)-dATP, N2-(p-n-butylphenyl)-dGTP), SH-blocking compounds (7). Inactivated by heating at 75C for 10 min. 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, 0.4mBq/ml [3H]-dTTP, 0.2mM heat-denatured and nuclease-digested calf thymus DNA. Endodeoxyribonuclease Assay: No detectable degradation was observed after incubation of supercoiled plasmid DNA with T4 DNA Polymerase. Protocol for blunting of 5- or 3-overhangs: 1. Prepare the following reaction mixture: 5X reaction buffer 4uL Linear DNA or PCR product 1ug dNTP Mix, 2mM each (R0241) 1 µL (0.1mM Final concentration) T4 DNA Polymerase 0.2uL (1U) Water, nuclease-free to 20uL 2. Mix thoroughly, spin briefly and incubate at 11C for 20 min or at room temperature for 5 min. 3. Stop the reaction by heating at 75C for 10 min. 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 37C. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. 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:
104
Form:
Supplied as a liquid in 20mM potassium phosphate, pH 7.5, 200mM potassium chloride, 2mM DTT, 50% glycerol.
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