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