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DESCRIPTION
T4 DNA Polymerase catalyzes
the 5'¡æ3' polymerization of DNA from a primed single-stranded DNA
template. This enzyme has a highly active 3'¡æ5' proofreading exonuclease
activity but lacks 5'¡æ3' exonuclease activity.
SOURCE
Recombinant strain of E. coli.
APPLICATIONS
¡Ü Filling-in or labeling
the recessed 3' termini created by restriction enzyme digestion of DNA.
¡Ü 3'-end labeling of DNA.
¡Ü Conversion of termini of
double-stranded DNA to blunt-ended molecules.
¡Ü Labeling DNA fragments
for use as hybridization probes.
UNIT DEFINITION
One unit is defined as
the amount of enzyme required to catalyze the incorporation of 10nmoles of
total nucleotide into an acid insouble form in 30 minutes at 37¡É.
STANDARD UNIT ASSAY CONDITIONS
10mM
Tris-HCl (pH7.9), 50mM NaCl, 10mM MgCl2, 1mM DTT, 33¥ìM dATP, dCTP and
dGTP, 33¥ìM [3H-dTTP], 70¥ìg/ml denatured calf thymus DNA, and 170¥ìg/ml BSA.
CONCENTRATION: 1-3 units/¥ìl.
10X REACTION BUFFER
100mM Tris-HCl
(pH7.9), 500mM NaCl, 100mM MgCl2, 10mM DTT. Supplement with 50¥ìg/ml BSA.
dNTPs not included.
STORAGE CONDITIONS
100mM Potassium
phosphate (pH6.5), 10mM 2-mercaptoethanol and 50% glycerol. Store at
-20¡É.
REFERENCES
1. Kunkel, T. A. et al.
(1987) Methods Enzymol. 154, 367.
2. Sambrook, J., Fritsch, E.F. and
Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor.
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