Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I. It exhibits 5'→3' polymerase activity and 3'→5' exonuclease (proofreading) activity, but lacks 5'→3' exonuclease activity of DNA polymerase I.
- Incorporates modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, biotin-, digoxigenin- and fluorescently-labeled nucleotides)
- Active in restriction enzyme, PCR, RT, and T4 DNA Ligase buffers
- DNA blunting by fill-in 5'-overhangs (see Reference 1)
- Random-primed DNA labeling (see References 2-4)
- Labeling by fill-in 5'-overhangs of dsDNA
- DNA sequencing by the Sanger method (see Reference 5)
- Site-specific mutagenesis of DNA with synthetic oligonucleotides (see Reference 6)
- Second strand synthesis of cDNA (see Reference 7)
10X Reaction Buffer 500mM Tris-HCl (pH8.0 at 25°C), 50mM MgCl2, 10mM DTT. Definition of Activity
- Unit One unit of the enzyme catalyzes the incorporation of 10nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30minutes at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.
- Enzyme activity is assayed in the following mixture: 67mM potassium phosphate (pH7.4), 6.7mM MgCl2, 1mM 2-mercaptoethanol, 0.033mM dATP, 0.033mM dTTP, 0.4MBq/mL [3H]-dTTP, and 62.5µg/mL poly(dA-dT)·poly(dA-dT).
Hazardous No Inactivation Inactivated by heating at 75°C for 10min or by addition of EDTA. Inhibition Inhibitors: metal chelators, PPi, Pi (at high concentrations) (see Reference 8). Molecular Weight 68kDa monomer. Quality Control
- The absence of endodeoxyribonucleases confirmed by appropriate quality test.
- Functionally tested for fill in of 5'-overhanging DNA termini and for random primed DNA labeling.
Source E. coli cells with a cloned fragment of the 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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