Thermo Scientific Klenow Fragment, exo-, is the large fragment of DNA polymerase I . It exhibits 5'→3' polymerase activity, but lacks the 3'→5' and 5'→3' exonuclease activities of DNA Polymerase I. The 3'→5' exonuclease activity of the enzyme is eliminated by mutations in the 3'→5'-exonuclease active site ( see Reference1).
- Lacks 3'→5' exonuclease activity
- Incorporates modified nucleotides (e.g., Cy3-, Cy5-, fluorescein-, rhodamine-, aminoallyl-, biotin-labeled nucleotides)
- Active in restriction enzyme, PCR, and RT buffers
- Random-primed DNA labeling ( see References 2-4)
- Labeling by fill-in 5'-overhangs of dsDNA
- Strand displacement amplification (SDA) ( see Reference 5)
- DNA sequencing by the Sanger method ( see Reference 6)
Klenow Fragment, exo- is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3'-terminus of blunt-end DNA substrates in a non-template directed fashion ( see Reference8).
|10X Reaction Buffer||500 mM Tris-HCl (pH8.0 at 25°C), 50mM MgCl 2 , 10mM DTT.|
|Definition of Activity Unit||
|Inactivation||Inactivated by heating at 75°C for 10min or by the addition of EDTA.|
|Inhibition||Inhibitors: metal chelators, PP i , P i (at high concentrations) ( see Reference 7).|
|Molecular Weight||68kDa monomer.|
|Source||E. coli cells with a cloned DNA fragment of the mutated polA gene.|
|Storage Buffer||The enzyme is supplied in:
25mM Tris-HCl (pH7.5), 0.1mM EDTA, 1mM DTT, and 50%(v/v) glycerol.
|Storage Condition||-20 C|
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