RNA Synthesis Protocol (M0255)
- Prepare the desired DNA template by PCR amplification. Use one primer that contains 18-22 nucleotide template specific sequence and an additional tail sequence at the 5´end (5´GGAAAAAAA-N(18-22)3´, where N(18-22) is the template-specific stretch in the primer). This will generate the required sequence in the RNA. The other primer should contain a T7 promoter sequence to be used in transcription (5´TAATACGACTCACTATAGG-N(18-22)3´, where N(18-22) is the template-specific stretch in the primer).
- Prepare RNA by in vitro transcription using T7 RNA Polymerase as described in the HiScribe Transcription Kit (NEB #E2000S ).
- Set up a dsRNA synthesis reaction using the following reaction conditions:
1X phi6 RdRp Reaction Buffer
1.5 mM MnCl2
20-100 ng/µl purified template RNA
0.1-0.2 mM ATP, CTP and UTP
0.3-0.6 mM GTP
- Add 1 unit of phi6 RNA Polymerase (RdRP) for a 40 µl reaction volume.
- Incubate at 32°C for 1-4 hours.
- Purify the amplified RNA using standard methods necessary for your downstream application.