Protein construct: Wild-type Human Mitochondrial RNA Polymerase purified from a baculovirus insect protein expression system.
MW: 130 kDa
Enzyme concentration: 7.5 µM
RNA polymerase assay: The RNA polymerase activity is measured by using the Human Mitochondrial RNA Polymerase Assay kit (Catalog No. MRPA100K).
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 Human Mitochondrial RNA Polymerase –for 100 assays (Catalog No. MRPA-100) includes 63 µl of 50 x Human Mitochondrial RNA Polymerase (7.5 µM). It is for 100 assays.
Assay Protocol using the Human Mitochondrial RNA Polymerase Assay kit
The following assay protocol is based on the 384-well plate assay format (plate type: Matrix 4318 or alike). The reaction volume is 30 µl and the final assay volume is 60 µl. For 96-well plate assays (plate type: Costar 3915 or alike), the reaction volume is 60 µl and the final assay volume is 120 µl.
1. Reagent preparation:
(1) 10 x DNA: Dilute the 100 x DNA 10-fold with water. Each assay uses 3 µl of 10 x DNA.
(2) 10 x enzyme: Dilute the 50 x Human Mitochondrial RNA Polymerase 5-fold with the 1 x assay buffer. Each assay uses 3 µl of 10 x enzyme.
(3) 10 x NTP mix: Dilute the 50 x NTP mix 5-fold with water. Each assay uses 3 µl of 10 x NTP mix.
(4) 1 x dye: Dilute the 10 x fluorescence dye 10-fold with water. Each assay uses 30 µl of 1 x dye.
2. Reaction:
The total volume of each reaction mixture is 30 µl including 18 µl of H2O, 3 µl of 10 x buffer, 3 µl of 10 x DNA template, 3 µl of 10 x enzyme, 3 µl of 10 x NTP mix. Incubate the reaction mixture at 37°C for 2 hours.
3. Detection:
Mix 30 µl of 1 x dye with 30 µl of the assay reaction mixture for 5 min and read the fluorescence at 535 nm (excitation at 485 nm).
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