T7 RNA in vitro transcription is useful for the enzymatic synthesis of specific RNA based on the DNA sequence downstream the appropriate promoter. Chemical modifications can be added accordingly into the RNA products when various modified rNTP building blocks are applied in the transcription.
5X Transcription Buffer: 200 mM Tris-HCl (pH 7.5), 30 mM MgCl2, 50 mM NaCl, and 10 mM spermidine. DTT and NTPs must also be added to the final reaction.
Standard Transcription Reaction:
Combine the following reaction components at room temperature in the order given:
Final Concentration
RNase-Free water ..................................................................................x μl
Linearized template DNA with appropriate promoter .......................0.5-1 μg ......................25-50 ng/μl
5X Transcription Buffer ......................................................................... 4 μl ..............................1X
10 mM ATP ............................................................................................1 μl ..............................0.5 mM
10 mM GTP ...........................................................................................1 μl ..............................0.5 mM
10 mM CTP ...........................................................................................1 μl...............................0.5 mM
10 mM UTP ............................................................................................1 μl................................0.5 mM
100 mM DTT .......................................................................................2 μl ............................. 10 mM
10 U T7 RNA Polymerase.......................................................................y μl ............................. 0.5 U/μl
Total reaction volume 20 μl.
Incubate at 37°C for 2-16 hours. After reaction, 1μl RNase-Free DNase I can be added and incubate for 15 minutes at 37°C to digest DNA template. The transcription product is desalted and PAGE- or HPLC- purified accordingly.