Klenow Fragment (Exo^-)Klenow Fragment (exo^-)

Source: Escherichia coli

Large fragment of E. coli DNA Polymerase l, which lacks both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities of Polymerase l.

Description:

• Devoided of both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities.
• Ultrapure recombinant enzyme.
• 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).

Unit Definition: One unit is the amount of enzyme required to incorporate 10 nmol of total nucleotide into acid‑insoluble form in 30 min at 37°C.

Storage Conditions: Store at –20^oC.

eStorage Buffer: 50 mM potassium phosphate, pH 7.0, 0.25 mM dithiothreitol and 50% glycrol.

Assay Conditions:  67 mM potassium phosphate, pH 7.4, 6.7 mM MgCl2, 1.0 mM dithiothreitol, 0.033 mM [a‑³²P]dATP, 0.033 mM dCTP, 0.033 mM dGTP, 0.033 mM dTTP and 4.5 µg activated DNA. Incubation is at 37°C for 30 min in a reaction volume of 100 µl.

Quality Control:

3'‑Exonuclease: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) and 5 pmoles of 3'‑ends of lambda/Taq I fragments (3'‑labeled with T4 DNA Polymerase and [³H]dCTP), incubated for 1 hr at 37°C resulted in ≤1.0 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.

5'‑Exonuclease/5'‑Phosphatase: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) with 0.25 µg of 5'‑ends of [5'‑³²P]lambda/Hae III fragments for 1 hr at 37°C resulted in ≤1.0 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.

Nicking: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) with 1µg of pBR322 DNA at 37°C for 1 hr resulted in ≤5% of RFI to RFII DNA. Reaction volume 50 µl.

Purity:  > 99% pure, as judged by SDS-polyacrylamide gel electrophoresis.

References:

1. 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.

2. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-546.7

3. Houdebine, L. M. (1976) Nucleic Acids Res. 3, 615-630.

Sambrook, J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, second edition pp. 5.34, 5.40-5.43 Cold Spring Harbor Laboratory, Cold Spring Harbor.

Large fragment of E. coli DNA Polymerase l, which lacks both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities of Polymerase l.

Description:

• Devoided of both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities.
• Ultrapure recombinant enzyme.
• 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).

Unit Definition: One unit is the amount of enzyme required to incorporate 10 nmol of total nucleotide into acid‑insoluble form in 30 min at 37°C.

Storage Conditions: Store at –20^oC.

eStorage Buffer: 50 mM potassium phosphate, pH 7.0, 0.25 mM dithiothreitol and 50% glycrol.

Assay Conditions:  67 mM potassium phosphate, pH 7.4, 6.7 mM MgCl2, 1.0 mM dithiothreitol, 0.033 mM [a‑³²P]dATP, 0.033 mM dCTP, 0.033 mM dGTP, 0.033 mM dTTP and 4.5 µg activated DNA. Incubation is at 37°C for 30 min in a reaction volume of 100 µl.

Quality Control:

3'‑Exonuclease: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) and 5 pmoles of 3'‑ends of lambda/Taq I fragments (3'‑labeled with T4 DNA Polymerase and [³H]dCTP), incubated for 1 hr at 37°C resulted in ≤1.0 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.

5'‑Exonuclease/5'‑Phosphatase: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) with 0.25 µg of 5'‑ends of [5'‑³²P]lambda/Hae III fragments for 1 hr at 37°C resulted in ≤1.0 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.

Nicking: Incubation of 5, 10, and 20 units of Klenow Fragment (Exonuclease Free) with 1µg of pBR322 DNA at 37°C for 1 hr resulted in ≤5% of RFI to RFII DNA. Reaction volume 50 µl.

Purity:  > 99% pure, as judged by SDS-polyacrylamide gel electrophoresis.

References:

1. 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.
2. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-546.7
3. Houdebine, L. M. (1976) Nucleic Acids Res. 3, 615-630.

Sambrook, J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, second edition pp. 5.34, 5.40-5.43 Cold Spring Harbor Laboratory, Cold Spring Harbor.