Protein construct: Wild-type S. pneumoniae DNA primase purified from a bacterial expression system.
MW: 68 kDa
Enzyme concentration: 10 µM
Enzyme activity assay: The DNA primase activity is measured by using the S. pneumoniae DNA Primase Assay Kit (Catalog No. PGA100K).
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 S. pneumoniae DNA Primase – for 100 assays (Catalog No. DNAG-100SP) includes 45 µl of 100 x S. pneumoniae primase. It is for 100 assays of S. pneumoniae DNA primase reactions in a 96-well plate format or 200 assays in 384-well assay format.
Enzymes and Substrates
Bacterial enzymes
Reference
Lacriola CJ et al, Inhibition of DNA replication in Staphylococcus aureus by tegaserod. The Journal of Antibiotics. 70, 918–920 (2017).
Assay Protocol using the S. pneumoniae DNA Primase Assay Kit
1. Reagent preparation:
10 x DNA: dilute the 100 x DNA with water.
10 x enzyme: Dilute the 100 x enzyme stock with the 1 x assay buffer to make the 10 x enzyme.
10 x NTP mix: dilute the 100 x NTP 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 40µl including 24µl of H2O, 4µl of 10 x Buffer, 4µl of 10 x DNA template, 4µl of 10 x enzyme, 4µl of 10 x NTP mix. Incubate the reaction mixture at 37°C for 60 min.
Note: The assay solution is composed of 10 mM MES, pH 6.5, 5 mM magnesium acetate, 12.5 mM ammonium chloride, 0.5 mM DTT, 5 mM calcium chloride, 0.003% Brij-35, 100 nM DNA, 0.5 mM NTPs, 100 nM enzyme.
3. Detection:
Add 80µl of the 1 x fluorescence dye into the 40µl 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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