Exonuclease III

#3005

 

4,000 units

#3005L

 

20,000 units

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
Generating nested sets of deletions of the terminal sequences of double-stranded linear DNAs.
Site-directed mutagenesis.
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.

STANDARD UNIT ASSAY CONDITIONS
50mM Tris-HCl (pH7.6), 1mM MgCl2, 1mM DTT and 125 g calf thymus DNA.

CONCENTRATION: 150-200 units/l.

10X REACTION BUFFER
500mM Tris-HCl (pH8.0@25), 50mM MgCl2, 10mM 2-mercaptoethanol.

STORAGE CONDITIONS
20mM Tris-HCl (pH7.9), 1mM DTT. 160mM KCl and 50% glycerol. Store at -20.

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.