1. DNA template preparation
Linearized plasmids with double-stranded T7 promoters or PCR amplification products can be used as T7 High Yield RNA Synthesis Kit in vitro transcription templates, which can be dissolved in TE buffer or RNase free H2O.
T7 promoter sequence: TAATACGACTCACTATAG*GG (Note: G* is the first base of RNA transcription).
A. Plasmid template
Insert the target DNA to the plasmid vector containing the T7 promoter, and then treated with restriction enzymes, purified after completely linearized.
Note:
1. The circular plasmids have no effective termination; RNA products of different lengths will be transcribed. In order to obtain a specific length RNA, the plasmid must be completely linearized.
2. The restriction enzyme selected for plasmid linearization needs to be on the right side of the promoter region, downstream of the inserted DNA fragment, and has no recognition site in the inserted DNA fragment. The restriction enzyme should be capable of forming 5' sticky ends or smooth ends.
3. In order to avoid the influence of protein and salt ions on the system, the plasmid is recommended to be purified when used as a template for in vitro transcription after linearization.
B. PCR product template
The PCR product with T7 promoter can be used as an in vitro transcription template. First, add the T7 promoter sequence (TAATACGA CTCACTATAGGG) to the 5' end of the upstream primer sense strand; next, the T7 promoter DNA template is amplified under the action of high-fidelity enzyme; then transcription is performed. PCR products can be used directly as templates without purification, but higher RNA output will be obtained after purification.
Note:
1. The specificity and concentration of the PCR product must be confirmed by electrophoresis when used as a template. Add 2-5 μL of PCR product into the 20 μL reaction system.
2. In order to obtain more high-quality RNA, the PCR product should be recovered by gel and used as a template for vitro transcription.

2. In vitro RNA transcription
A. Thawing reagents
The T7 RNA polymerase and RNase Inhibitor were centrifuged briefly and placed on ice. Thaw 10× Transcription Buffer and ribonucleotides (ATP, CTP, GTP, UTP), mix and centrifuge to the bottom of the tube, place 10× Transcription Buffer at room temperature, and place 4 types of ribonucleotides on ice.
B. Assembly transcription reaction at room temperature
Prepare the reaction system according to the following system:

C. Incubate at 37°C for 2 hours
Mix the above reaction solution, briefly centrifuge to the bottom of the tube, and incubate at 37°C for 2h. If the transcript length is less than 100 nt, increase the reaction time to 4-8h.
D. DNase I treatment (optional)
After the reaction is complete, add 2 μl of DNase I (RNase free) to each tube and incubate at 37°C for 30 mins to remove the template DNA.

3. Product purification
After electrophoretic analysis and purification, the synthesized RNA can be used for downstream experiments.

1. As spermine components are contained in 10×Transcription Buffer, it will precipitate with template DNA at low temperature, so preparation of reaction solution should be carried out at room temperature, the order of component loading should be adjusted, the system should be calculated, and water, buffer and NTP should be added first, and template and enzyme should be added finally.

2. Perform the reaction in a PCR machine with the hot lid open to prevent the reaction solution from evaporating for a long time.

3. The reaction product may have a white precipitate. This is free pyrophosphate and magnesium ions produce the magnesium pyrophosphate in the reaction, won’t affect the subsequent experiments. You can add some EDTA to clear it. If the addition of EDTA affects subsequent experiments, the supernatant can also be recovered by centrifugation.

4. The reagents and containers should be without RNase contamination.