|
DESCRIPTION
Exonuclease III catalyzes the stepwise removal of 5' mononucleotides from
the 3'-hydroxyl termini of double-stranded DNA. Linear double-stranded
DNA and circular DNAs containing nicks or gaps are substrates. The enzyme
is not active on single-stranded DNA or double-stranded DNA with a protruding
3' terminus. It will not degrade double-stranded DNA with 3' overhang
of four bases or longer. The enzyme also carries three other activities:
an endonuclease specific for apurinic DNA, an RNase H activity, and a
3' phosphatase activity.
SOURCE
Recombinant E. coli strain.
APPLICATIONS
¡Ü Generating nested sets
of deletions of the terminal sequences of double-stranded linear DNAs.
¡Ü Site-directed mutagenesis.
¡Ü Preparation of single-stranded
substrates for dideoxy sequencing.
UNIT DEFINITION
One unit is defined as
the amount of enzyme required to produce 1nmole of acid-soluble
nucleotides in 30 minutes at 37¡É.
STANDARD UNIT ASSAY CONDITIONS
50mM
Tris-HCl (pH7.6), 1mM MgCl2, 1mM DTT and 125¥ì g calf thymus DNA.
CONCENTRATION: 150-200 units/¥ìl.
10X REACTION BUFFER
500mM Tris-HCl
(pH8.0@25¡É), 50mM MgCl2, 10mM 2-mercaptoethanol.
STORAGE CONDITIONS
20mM Tris-HCl
(pH7.9), 1mM DTT. 160mM KCl and 50% glycerol. Store at -20¡É.¡¡
REFERENCES
1. Rogers, S.G. and Weiss,
B. (1980) Methods. Enzymol. 65, 201.
2. Weiss, B. (1976) J. Biol.
Chem. 251, 1896.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, second ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor.
|