Plasmid templates were designed using Geneious software, and codon optimized and ordered from GenScript. Plasmids were linearized with Not‐I HF (New England Biolabs) overnight at 37 °C. Linearized templates were purified by sodium acetate (Thermo Fisher Scientific) precipitation and rehydrated with nuclease‐free water. In vitro transcription was performed overnight at 37 °C using a HiScribe T7 Kit (NEB) following the manufacturer's instructions (complete N1‐methyl‐pseudouridine modification). The resulting RNA was treated with DNase I (Aldevron) for 30 min and purified using lithium chloride precipitation (Thermo Fisher Scientific). The RNA was heat denatured at 65 °C for 10 min before capping with a Cap‐1 structure using guanylyl transferase and 2′‐O‐methyltransferase (Aldevron). mRNA was then purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB), and purified again. mRNA concentration was measured using a Nanodrop. mRNA stock concentrations were 1–3 mg mL−1 and were stored at −80 °C until use. Purified mRNA products were analyzed by gel electrophoresis to ensure purity.
Dnase 1
Manufactured by Aldevron
DNase I is a laboratory enzyme that catalyzes the hydrolysis of DNA, breaking down DNA molecules into smaller fragments. It is commonly used in various molecular biology and biochemistry applications to remove unwanted DNA from samples.
Automatically generated - may contain errors
Lab products found in correlation
Guanylyl transferase, by Aldevron (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Ribogreen assay, by Thermo Fisher Scientific (2 mentions)
Pyrophosphatase enzymes, by Aldevron (2 mentions)
Capto core 700 resin, by GE Healthcare (2 mentions)
Rnase inhibitor, by Aldevron (2 mentions)
S adenosyl methionine, by Aldevron (2 mentions)
Hiscribe t7 kit, by New England Biolabs (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
Noti hf, by New England Biolabs (1 mentions)
Alkaline phosphatase, by New England Biolabs (1 mentions)
T7 polymerase, by Aldevron (1 mentions)
Rntp mix, by New England Biolabs (1 mentions)
Capto, by Cytiva (1 mentions)
3 protocols using dnase 1
1
Protocol for Optimized mRNA Production
2
Generation of Stabilized mRNA Stocks
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Generation of saRNA stocks was achieved by T7 promoter-mediated in vitro transcription using NotI-linearized DNA template. In vitro transcription was performed using an in-house-optimized protocol using T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes procured from Aldevron. DNA plasmid was digested away (DNase I, Aldevron) and cap0 structures were added to the transcripts by vaccinia capping enzyme, GTP, and S-adenosyl-methionine (Aldevron). RNA was then purified from the transcription and capping reaction components by chromatography using a CaptoCore 700 resin (GE Healthcare) followed by diafiltration and concentration using tangential flow filtration. The saRNA material was terminally filtered with a 0.22 μm polyethersulfone filter and stored at −80°C until use. All saRNA was characterized by agarose gel electrophoresis and quantified both by UV absorbance (NanoDrop 1000) and Ribogreen assay (Thermo Fisher). OVA-expressing mRNA was obtained from a commercial vendor (TriLink CleanCap OVA mRNA, L-7610).
3
In Vitro Transcription of saRNA
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Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using NotIlinearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified using Capto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). Terminal filtration of the saRNA material was done using a 0.22 µm PES filter, and the saRNA materials were stored at -80°C until use/complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity, and RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).
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