We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA). The recommended ratio of cap analog to GTP is 4:1. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts; however, it also significantly decreases the yield of the reaction. Cap analogs are sold separately. Please refer to the companion products section.
- 1．Prepare 40 mM cap analog. Cap analog (ARCA, SV2000) is supplied in a lyophilized form of 1 μmol per tube. Dissolving it in 25 μl nuclease-free water will yield a concentration of 40 mM.
- 2．Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to the bottoms of tubes.
- 3．Assemble the reaction at room temperature in the following order:
| Nuclease-free water | X μl |
| NTP Buffer Mix | 2 μl (2 mM each NTP final) |
| Cap Analog (40 mM) | 4 μl (8 mM final) |
| Template DNA | X μl (1μg) |
| T7 RNA Polymerase Mix | 2 μl |
| Total reaction volume | 20 μl |
- 4．Mix thoroughly, pulse-spin and incubate at 37℃ for 2 hours. The yield per reaction is 30–40 μg RNA with approximately 80% capped RNA transcripts. Figure 1 shows the time course of capped RNA synthesis from 1 μg control template. Most reactions will be complete in 1 hour.
Figure 1. Capped RNA Synthesis with ARCA
Reactions were incubated at 37℃ in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.
- 5．Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37℃ for 15 minutes.
- 6．Proceed with purification of synthesized RNA or analysis of transcription products by gel electrophoresis.
