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.
Megascript reaction
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The MEGAScript reaction is a high-yield in vitro transcription system used to generate large quantities of RNA from DNA templates. It provides efficient and reproducible RNA synthesis to support a variety of RNA-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Ultrapure h2o, by Thermo Fisher Scientific (4 mentions)
Scriptcap m7g capping system, by CellScript (3 mentions)
Scriptcap 2 0 methyltransferase kit, by CellScript (3 mentions)
Plasmid plus maxiprep kit, by Qiagen (2 mentions)
Carbenicillin, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Scriptcap, by CellScript (1 mentions)
5 protocols using megascript reaction
1
Uncapped RNA Capping and Purification
2
Synthesis of Post-Transcriptionally Capped saRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
4
Plasmid-based saRNA Production and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmid encoding saRNA construct was transformed into E. coli (institute Pasteur, Iran), cultured in Luria Broth with 100 μg/mL carbenicillin (Sigma Aldrich, UK). Plasmids were purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and their concentration and purity were measured on a NanoDrop spectrophotometer (ThermoFisher, UK). Then, cloned plasmids were linearized using MluI for 3 h at 37 °C. Then, saRNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 1 h at 37 °C. One μg linear saRNA was mixed with 1 μM ScriptCap™ (CellScript, WI, USA) for 1 h at 37 °C. Synthesized saRNA was purified by LiCl precipitation, re-suspended in RNA storage buffer, and stored at − 80 °C. To evaluate the purity of synthesized saRNA, the A260/A280 ratio was measured using the NanoDrop spectrophotometer (ThermoFisher, UK) [21] (link).
5
SARS-CoV-2 Stabilized Pre-Fusion RNA Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Self-amplifying RNA encoding the pre-fusion stabilized SARS-CoV-2 was produced using in vitro transcription. pDNA was transformed into E. coli (New England BioLabs, UK), cultured in 100 mL of Luria Broth (LB) with 100 μg mL−1 carbenicillin (Sigma Aldrich, UK). Plasmid was purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and the concentration and purity was measured on a NanoDrop One (ThermoFisher, UK). pDNA was linearized using MluI for 3 h at 37 °C. Uncapped in vitro RNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 2 h at 37 °C, according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20 °C, centrifuged at 14,000 RPM for 20 min at 4 °C to pellet, washed with 70% EtOH, centrifuged at 14,000 RPM for 5 min at 4 °C and resuspended in UltraPure H2O (Ambion, UK). Purified transcripts were capped using the ScriptCap™ Cap 1 Capping System Kit (CellScript, WI, USA) for 2 h at 37 °C, according to the manufacturer’s protocol. Capped transcripts were purified by LiCl precipitation as described above, resuspended in RNA storage buffer (10 mM HEPES, 0.1 mM EDTA, and 100 mg mL−1 trehalose) and stored at −80 °C until further use.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!