Protocol for T7 Phage RNA Polymerase

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T7, T3, and SP6 Phage RNA Polymerases Related Products: The following products are also available: – AmpliScribe T7-Flash Transcription Kit – NTP Solutions – DuraScribe® T7 Transcription Kit – RNase-Free DNase I Standard Transcription Reaction: Synthesis of Nonradioactive RNA 1. Combine the following reaction components at room temperature in the order given: Final Concentration x μl RNase-Free water --- 0.5-1 μg linearized template DNA with appropriate promoter..........25-50 ng/μl 4 μl 5X Transcription Buffer.......................................................................................1X 1 μl 10 mM ATP....................................................................................................0.5 mM 1 μl 10 mM CTP...................................................................................................0.5 mM 1 μl 10 mM GTP...................................................................................................0.5 mM 1 μl 10 mM UTP...................................................................................................0.5 mM 2 μl 100 mM DTT..................................................................................................10 mM 10 U T7 Phage RNA Polymerase..................................................................... 0.5 U/μl 20 μl Total reaction volume 2. Incubate at 37°C for 2 hours. 3. (Optional) Add 1 μl (1 MBU) of RNase-Free DNase I and incubate for 15 minutes at 37°C. The volume of this reaction may be increased or decreased proportionately. Synthesis of Radioactive RNA: Radioactive RNA may be synthesized by adding a radioactive nucleoside triphosphate and decreasing the final concentration of the corresponding nonradioactive NTP to 15 μM. As the total concentration of the labeled NTP will be limiting, virtually all of the radioactivity will be incorporated into RNA. For example, to label with α-[ 32 P]-CTP, first prepare a stock of 100 μM CTP by diluting the 10 mM CTP stock 1 to 100 with RNase-Free H 2 O. Then add 3 μl of the 100 μM CTP and the desired amount of α-[ 32 P]-CTP in place of the 10 mM CTP and some of the water in the above protocol. DuraScribe is a registered trademark of, and AmpliScribe, and T7-Flash are trademarks of Epicentre, Madison, Wisconsin. Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania. Visit our technical blog: epicentral.blogspot.com 2 www.epicentre.com
T7, T3, and SP6 Phage RNA Polymerases techhelp@epicentre.com • (800) 284-8474 3
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Protocol for T7 Phage RNA Polymerase

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