Exonuclease-Free Klenow
(Deoxynucleoside triphosphate: DNA Deoxynucleosidyltransferase, Large Fragment of DNA Polymerase I, Exonuclease-Free Klenow Enzyme, E.C. 2.7.7.7)
Source:
E. coli strain containing an overproducing clone of E. coli Exonuclease-free Klenow fragment.
Description:
The large fragment of DNA polymerase I from E. coli, Klenow, has been genetically engineered to eliminate the 3'→5' exonuclease activity normally associated with this polymerase(1). Exo-free Klenow is useful in applications where accompanying exonuclease activity is of significant concern, such as random primed labeling, DNA sequencing by the Sanger, et al. method, or strand displacement amplification (SDA).
Properties:
Molecular Weight: 68 kDa
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating double- and single-stranded endonucleases and exonucleases.
Storage Buffer:
50mM potassium phosphate (pH 7.0), 1.0mM DTT, 1.0mM EDTA, 50% glycerol.
Assay Conditions:
The reaction mixture contains 100mM potassium phosphate (pH 7.4), 6.7mM MgCl2, 1mM DTT, 20μM poly(dA-dT)-(dA-dT), 33μM dATP, 33μM dTTP and enzyme. Incubation is for 30 min at 37°C.
Unit Definition:
One unit is the amount of enzyme required to catalyze the incorporation of 10 nmol of total deoxyribonucleotides into an acid-precipitable material in 30 min at 37°C.
Concentration:
10 units/μl
Tested User Friendly™ Functional Test:
Filling-in 3' recessed ends with >50% incorporation of radiolabeled dATP into 0.1-4 μg of restricted DNA in 15 min at 30°C.
Functionally Tested 10X Exonuclease-Free Klenow Reaction Buffer (1 ml included, PN 70095):
0.5M Tris-HCl (pH 7.4), 0.1M MgCl2, 10mM DTT.
References:
- DERBYSHIRE, V., FREEMONT, P. S., SANDERSON, M. R., BEESE, L., FRIEDMAN, J. M., JOYCE, C. M. AND STEITZ, T. A. (1988) Science 240, 199-201.
- SANGER, F., NICKLEN, S. AND COULSEN, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467.
- WALKER, G. T. (1993) PCR Methods Appl. 3, 1-6.
- CLARK, J. M., JOYCE, C. M. AND BEARDSLEY, G. P. (1987) J. Mol. Biol. 198,
123-127.
Applications:
- Random primed DNA labeling.
- Strand displacement amplification (SDA)(3).
- DNA sequencing by the Sanger dideoxy method(2).
- Labeling recessed 3' termini of DNA fragments with labeled dNTP.
Note: Exonuclease-Free Klenow is not recommended for fill-in reactions before DNA ligation, since it frequently adds one or more extra nucleotides to the 3'-terminus of a blunt-ended DNA substrate in a non-template directed fashion(4).
Storage:
Shipped on dry ice. Store at -20°C.