| Solution Information | help | |
| Enzyme: | DNA-directed RNA polymerase subunit beta | |
| inhibitor: | BDBM41208 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials The synthesis of Um-pppp-G and the purification of E. coli RNA polymerase and PUC19 plasmid DNA were carried out by Mustaev and co-workers as previously described (Koslov M., et al. Anal. Biochem. 342 (2005) 206-213). ATP, CTP, and UTP were purchased from Roche Applied Science; alkaline phosphatase was from New England Biolabs (Cat #M0290S); buffers and other reagents were from Sigma. Low-volume 384-well black plates were from Corning (Item #3676). Stock solutions were made up as follows and stored at -80 C: (1)Um-pppp-G: 12 mM in water (2)NTP mix: 25 mM each of ATP, CTP, and UTP in water (3)DNA template: 2 mg/mL of PUC19 (4)RNA polymerase: 3 mg/mL (5)Transcription assay buffer (10x): a.HEPES, pH 8.0 (200 mM) b.NaCl (1 M) c.Magnesium chloride (100 mM) d.Manganese chloride (15 mM) e.EDTA (1 mM) Alkaline phosphatase was stored as supplied by the vendor at -20 C. AMPSO buffer, pH 9.2 (250 mM) was stored at room temperature. Assay RNA polymerase (20 ug/mL) was incubated | |
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