DNA Polymerase I Klenow Fragment, Exo-Minus, Recombinant
|Cloning||Storage: -20°CShipping: Blue Ice|
Klenow fragment, exo, is an N-terminal truncation of E. coli DNA Polymerase I at amino acid #323 . In this construct, the domain containing the 5´-3´ exonuclease activity is deleted, leaving the polymerase function intact. In addition two mutations are introduced (D355A and E357A) which abolish the 3´-5´ exonuclease activity. The enzyme features strand displacement capability , an error rate of @ 100x100e-6 bases and has no intrinsic exonuclease activity.
• Recombinant single protein; not a fusion construct.
• Generates probes using direct or indirect labeling methods.
• Specific Activity: 15,000-20,000 units/mg.
• Low DNA content
• High concentration, automation grade.
• Supplied with 10X Reaction Buffer. High concentrations supplied with 1X Dilution Buffer
Size1 100ul 5u/ul
Size2 50ul 40u/ul
Size3 20ul 200u/ul
Also included are 1ml dilution buffer and 2ml 10X reaction buffer.
Application(s): Dideoxy sequencing (2), blunt ending restriction fragments, second strand cDNA synthesis for labeling and for use in mutagenesis protocols (3). The enzyme is extensively utilized for labeling of DNA with nucleotide analogs for use as microarray probes (4).
10X Reaction Buffer: 500mM KPO4 pH 7.0, 60mM MgCl2, 50mM 2-mercaptoethanol
1X Dilution Buffer: 50mM KPO4 pH 7.0, 100mM KCl, 1mM DTT, 50% v/v glycerol
ds endonuclease (Degrade X174 RF): None Detected
ss endonuclease (Degrade M13mp18): None Detected
5' ss+ds exonuclease (Removal of labeled nucleotide from 5' end of a ss or ds oligonucleotide): None Detected
3' ss+ds exonuclease (Removal of labeled nucleotide from 3' end of a ss or ds oligonucleotide): None Detected
unit functional assay:
1 unit incorporates 10nMol32P dTTP into acid insoluble material in 30 minutes at 37°C using poly dA/dT as substrate
Storage and Stability:
For long-term storage, store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Source: E. coli
Concentration: As reported
Form: 50mM KPO4 pH 7.0, 100mM KCl, 1mM DTT, 50% v/v glycerol
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
1. Derbyshire, V., et al., Science 240: 199-201 (1988). 2. Feinberg, A.P. and B. Vogelstein, Anal. Biochem. 137: 266-267 (1984). 3. Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977). 4. Walker, G.T., PCR Methods Appl. 3: 1-6 (1993). 5. Clark, J.M., et al., J. Mol. Biol. 198: 123-127 (1987).
6. Gubler, U. (1987) S.L. Berger and A.R. Kimmel (Eds.), Methods in Enzymology, 152, pp. 330-335. San Diego: Academic Press. 7. Joseph Gilbert, Jeremy Hasseman, Robin Cline,Kathy Munoz, Jon Hnath Microbial Genomic DNA aminoallyl labeling for microarrays. S.O.P. from The Institute for Genomic Research (TIGR).