Applications
- Conversion of both 5´- and 3´-protruding DNA termini to blunt
ends.
- Cloning of PCR fragments. Treatment of PCR products containing 3´-A
overhangs with T4 DNA Polymerase and dNTPs produces blunt ends, which greatly
increases cloning efficiencies.1
- Production of site-specific mutations. Because T4 DNA Polymerase does
not displace oligonucleotides that are hybridized to DNA, it can be used
for site-specific mutagenesis by primer extension of "mutated" oligonucleotides
hybridized to ssDNA templates.2
- Labeling of 3´ termini of DNA molecules and synthesis of strand-specific
probes using the exonuclease and polymerase activities of T4 DNA Polymerase.2,3
T4 DNA Polymerase has both a DNA-dependent DNA polymerase activity and
a potent 3´→5´ exonuclease activity. These characteristics
make the enzyme useful for several molecular biology applications.
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.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5),
0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.
T4 DNA Polymerase 10X Reaction Buffer: 330 mM Tris-acetate (pH 7.8),
660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT. dNTPs
are not included in the 10X Reaction Buffer but are available separately.
Quality Control: T4 DNA Polymerase is tested for DNA synthesis and
is free of detectable endonuclease and RNase activities. |
Figure 1. Exonuclease and polymerase fill-in activities of T4 DNA Polymerase. Hind
III-digested lambda DNA was incubated with T4 DNA Polymerase in the absence
of dNTPs for the indicated time (min) at 37°C (lanes labeled with minus
sign). Note the progressive decrease in fragment size with increasing incubation
time. After exonucleolytic treatment for different time periods, aliquots
of each reaction were taken and dNTPs were added to 200 µM each. The
reactions were then continued for 10 minutes to allow the polymerase fill-in
reaction to occur, thus regenerating the original DNA fragments (lanes labeled
with plus sign). |
