Klenow Fragment, Exonuclease Free (Exo-)

Source

Escherichia coli

Description

• Lacks both 3'→5' exonuclease and 5'→3' exonuclease activities (1)
• Ultrapure recombinant enzyme

Applications

• Used for Sanger dideoxy sequencing (2)
• Suitable for second strand cDNA synthesis (3)
• Used for 3'-end labeling and filling in 5'-protruding sticky ends (4)

Reagents Supplied

10X Klenow Fragment (Exo-) Reaction Buffer

Unit Definition

One unit is the amount of enzyme required to incorporate 10 nmoles of total nucleotide into acid insoluble form at 37°C for 30 minutes

Heat Inactivation

75ºC for 15 minutes

Concentration

5 - 10 units/µl

Storage Buffer

50 mM potassium phosphate (pH 7.0)
0.25 mM dithiothreitol
50% glycerol

Assay Conditions

67 mM potassium phosphate (pH 7.4)
6.7 mM MgCl₂
1 mM dithiothreitol
0.033 mM of each dCTP, dGTP, dTTP and [α-³²P]dATP
4.5 µg activated DNA
Incubation is for 30 minutes at 37°C in a total reaction volume of 
100 µl

Quality Control

All preparations are assayed for contaminating 3'- and 5'-exonuclease and endonuclease activities

Storage Conditions

Store at -20°C
Product shipped on dry ice