Briefly, uncapped RNA was prepared using 1 μg of linearized DNA template in a MEGAScript reaction (Ambion, UK) according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20°C, pelleted by centrifugation at 14 000 RPM for 20 min at 4°C, washed once with 70% ethanol, centrifuged at 14 000 RPM for 5 min at 4°C and then resuspended in UltraPure H2O (Ambion). Purified transcripts were then capped using the ScriptCap m7G capping system (CellScript, Madison, WI, USA) and ScriptCap 2′-0-methyltransferase kit (CellScript) simultaneously according to the manufacturer’s protocol. Capped transcripts were then purified by LiCl precipitation, as detailed above, resuspended in UltraPure H2O and stored at −80°C until formulation.
Scriptcap 2 0 methyltransferase kit
Manufactured by CellScript
Sourced in United States
The ScriptCap 2′-0-methyltransferase kit is a laboratory tool used in the capping process of in vitro transcribed mRNA. It catalyzes the methylation of the 2'-hydroxyl group of the first transcribed nucleotide, thereby creating the cap structure essential for mRNA stability and translation.
Automatically generated - may contain errors
3 protocols using scriptcap 2 0 methyltransferase kit
1
Uncapped RNA Capping and Purification
2
Synthesis of Post-Transcriptionally Capped saRNA
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Post-transcriptionally capped saRNA was synthesized as previously described [11 (link)]. Briefly, uncapped RNA was prepared using 1 μg of linearized DNA template in a MEGAScript reaction (Ambion, UK) according to the manufacturer's protocol. Transcripts were then purified by overnight LiCl precipitation at −20 °C, pelleted by centrifugation at 14,000 RPM for 20 min at 4 °C, washed once with 70% ethanol, centrifuged at 14,000 RPM for 5 min at 4 °C and then resuspended in UltraPure H2O (Ambion, UK). Purified transcripts were then capped using the ScriptCap m7G capping system (CellScript, Madison, WI, USA) and ScriptCap 2′-0-methyltransferase kit (CellScript) simultaneously according to the manufacturer's protocol. Capped transcripts were then purified by LiCl precipitation, as detailed above, resuspended in UltraPure H2O and stored at −80 °C until formulation.
3
Capped RNA Synthesis and Purification
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Briefly, uncapped RNA was prepared using 1 μg of linearized DNA template in a MEGAScript reaction (Ambion, UK) according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20°C, pelleted by centrifugation at 14,000 RPM for 20 min at 4°C, washed once with 70% ethanol, centrifuged at 14,000 RPM for 5 min at 4°C, and then resuspended in UltraPure H2O (Ambion, UK). Purified transcripts were then capped using the ScriptCap m7G capping system (CellScript, Madison, WI, USA) and ScriptCap 2′-0-methyltransferase kit (CellScript) simultaneously according to the manufacturer’s protocol. Capped transcripts were then purified by LiCl precipitation, as detailed above, resuspended in UltraPure H2O, and stored at −80°C until formulation.
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