Thermo Fisher Scientific Conventional in vitro Transcription User guide
- Type
- User guide
Protocol
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More than 10 µg of RNA transcript can be generated per
1 µg template DNA using the following protocol. The
reaction can be scaled up or down. For high yield
transcription, generating up to 200 µg RNA, use Tran-
scriptAid™ T7 High Yield Transcription Kit.
• Thaw frozen reagents, mix and centrifuge briey.
• Keep enzymes and nucleotides on ice.
• Keep the Reaction Buffer at room temperature.
1. Prepare the following reaction mixture at room
temperature:
5X Transcription buffer 10 µl
ATP/GTP/CTP/UTP Mix, 10 mM each 10 µl (2 mM nal
concentration)
Linearized template DNA 1 µg
RiboLock™ RNase Inhibitor 1.25 µl (50 u)
T7/T3/SP6 RNA Polymerase 1.5 µl (30 u)
DEPC-treated Water to 50 µl
Total volume 50 µl
2. Incubate at 37°C for 2 hours.
3. Optional: To remove template DNA add 2 µl (2 u) of
DNase I, RNase-free, mix and incubate at 37°C for
15 min.
4. Stop the reaction by addition of 2 µl 0.5 M EDTA,
pH 8.0 and incubate at 65°C for 10 min.
Note
RNA hydrolyzes if heated in the absence of a chelating agent.
Conventional in vitro Transcription
This protocol is for the Conventional in vitro Transcription
-
1
Thermo Fisher Scientific Conventional in vitro Transcription User guide
- Type
- User guide
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