DNA Polymerase T4

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 3'→5' 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 37°C.

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 75°C 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 11°C for 20 min or at room temperature for 5 min.
3. Stop the reaction by heating at 75°C 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 37°C.

Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt 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.