Protein construct: Wild-type E. coli DNA Polymerase III Alpha purified from a bacterial expression system.
MW: 94 kDa
Enzyme concentration: 2 µM
Enzyme activity assay: The DNA polymerase activity is measured by using the DNA Polymerase III Alpha Assay Kit (Catalog No. DPA100K)
Storage temperature: -20 or -80°C. Do not freeze-and-thaw repeatedly.
Enzyme dilution: Use the 1 x assay to dilute the enzyme just before the assay. Do not store diluted enzyme solution.
The E. coli DNA Polymerase III Alpha – for 100 assays (Catalog No. DPA-100EC) includes 33 µl of 100 x E. coli DNA polymerase III alpha. It is for 100 assays.
Assay Protocol using the DNA Polymerase III Alpha Assay Kit
1. Reagent preparation:
10 x DNA: dilute the 100 x DNA with water.
10 x enzyme: prepare 100 x DNA polymerase using the 1 x buffer for dilution.
10 x dNTP mix: dilute the 100 x dNTP mix 10-fold with water.
1 x fluorescence dye: dilute the 10 x fluorescence dye 10-fold with water.
2. Reaction:
The total volume of each reaction mixture is 30µl including 18µl of H2O, 3µl of 10 x Buffer DP, 3µl of 10 x DNA, 3µl of 10 x enzyme, 3µl of 10 x dNTP mix. Incubate the reaction mixture at 37°C for 60 min.
Note: The final concentrations are 20 mM HEPES, pH 7.5, 10 mM Mg(OAc)2 , 1 mM DTT, 30 nM DNA, 20 nM DNA polymerase III alpha subunit, 0.1 mM dATP, 0.1 mM dGTP.
3. Detection:
Add 30µl of the 1 x fluorescence dye into the 30µl of the reaction mixture. Incubate for 5 min. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
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