DNA Polymerase I

  Large (Klenow) Fragmen

#3004

 

150 units

#3004L

 

750 units

DESCRIPTION
Klenow Fragment is a proteolytic product of intact DNA polymerase I which retains 5'3' DNA polymerase and 3'5' exonuclease activity but lacks 5'3' exonuclease activity.

SOURCE
Recombinant E. coli strain.

APPLICATIONS
DNA sequencing by the Sanger dideoxy method.
Filling-in and/or labeling of recessed 3'-ends.
Second strand cDNA synthesis.
End-labeling of DNA molecules with protruding 3' tails.
Synthesis of dsDNA from ssDNA templates during in vitro mutagenesis.

UNIT DEFINITION
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of dNTPs into acid-insoluble form in 30 minutes at 37.

STANDARD UNIT ASSAY CONDITIONS
67mM KPO4 (pH7.5), 6.7mM MgCl2, 1mM DTT, 133M activated calf thymus DNA and 33M dATP, dCTP, dGTP and [3H]-dTTP.

CONCENTRATION : 5-10 units/l.

10X REACTION BUFFER
100mM Tris-HCl (pH7.5@37), 50mM MgCl2, 7.5mM DTT. dNTPs not included.

STORAGE CONDITIONS
50mM Tris-HCl (pH7.5), 1mM DTT, 0.1mM EDTA and 50% glycerol. Store at -20.

REFERENCES
1. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463.
2. Joyce, D.M. and Grindley, N.D.F. (1983) Proc. Natl. Acad. Sci. USA 80, 1830.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual,
    second ed., Cold Spring Harbor Laboratory, Cold Spring Harbor.
4. Gubler, U. (1987) Methods Enzymol. 152, 330.