Large Fragment of DNA Polymerase I, 3'→5' exo-
recombinant, E. coli
For in vitro use only!
Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into acid-insoluble material in 30 minutes at 37 °C.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Purity: > 95 % (SDS-PAGE)
Form: liquid (Supplied in 100 mM KH2PO4/K2HPO4 pH 6.5, 1 mM DTT and 50 % [v/v] glycerol)
Concentration: 10 units/μl
Klenow Fragment, 3'→5' exo- is a genetically engineered product of E. coli DNA polymerase I that catalyzes the 5'→3' synthesis of DNA but lacks its 3'→5' and 5'→3' exonuclease activities. The enzyme is recommended for second strand synthesis, particularly for incorporation of fluorescently labeled nucleotide analogs into DNA as well as DNA sequencing by the Sanger dideoxy method.
The polymerase activity is assayed in 1x Klenow Fragment Reaction Buffer including 300 μM dNTPs, M13mp18 ss-DNA and M13 universal primer. Quantification of ds-DNA is performed using PicoGreen®.
10x Reaction Buffer:
100 mM Tris-HCl pH 7.5, 50 mM MgCl2 and 75 mM DTT.
Absence of contaminants:
Tested for the absence of endo- and exodeoxy-ribonucleases.
20 minutes at 75 °C
Brakmann et al. (2001) High-Density Labeling of DNA: Preparation and Characterization of the Target Material for Single-Molecule Sequencing. Angew. Chem. Int. Ed. 40:1427.
Tveit et al. (2001) Fluorescence-based DNA polymerase assay. Anal. Biochem. 289:96.
Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463.
Pico Green® dsDNA quantitation reagent is a trademark of Molecular Probes, Inc.