Editing Cleancap mRNA Invitro Transcription (section)

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Nonobody mRNA</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">HiScribe T7 ARCA mRNA kit (with tailing; NEB; #E2060S). PCR product should be purified and run on an agarose gel to confirm amplicon size prior to its use as a template in the T7 ARCA mRNA transcription reaction. Generally, 0.1–0.5 mg of PCR fragments can be used in a 20-mL in vitro transcription reaction. After the preparation of PCR templates, standard mRNA synthesis with modified nucleotides should be started with thawing the necessary kit components, mixing, and pulse spinning in a microfuge to collect solutions to the bottoms of the tubes. The reaction was assembled at room temperature in the following order: nuclease-free water (to 20 uL), 2 x ARCA/NTP mix (10 uL), 5mCTP (10 mM, 2.5 uL), pseudo-UTP (10 mM, 2.5 uL), template DNA (1 mg), and T7 RNA polymerase mix (2 uL); mixed thoroughly; and pulse spun in a microfuge. Incubation was at 37oC for 30 min. DNase treatment to remove template DNA included adding 2 uL of DNase I, mixing well, and incubating at 37 oC for 15 min. poly(A) tailing starts with setting up the tailing reaction as below. Standard tailing reaction volume was 50 uL: H2O (20 uL), IVT reaction (20 uL), 10 X poly(A) polymerase reaction buffer (5 uL), and poly(A) polymerase. It was mixed thoroughly, pulse spun in a microfuge, and incubated at 37oC for 30 min. Synthesized mRNA can be purified by LiCl precipitation, phenol/chloroform extraction, followed by ethanol precipitation. To the 50-mL tailing reaction, 25 mL LiCl solution was added and mixed well. Incubation at -20 oC for 30 min was completed, as was centrifugation at 4oC for 15 min at top speed to pellet the RNA. The supernatant was removed carefully, the pellet was rinsed by adding 500 mL of cold 70% ethanol and centrifuged at 4oC for 10 min, ethanol was removed carefully, the tube was spun briefly to bring down any liquid on the wall, residual liquid was removed carefully using a sharp tip, the pellet was air dried, the mRNA was resuspend in 50 mL of 0.1 mM EDTA or a suitable RNA storage solution, and the RNA was heated at 65oC for 5– 10 min to completely dissolve the RNA and mixed well.</span>