MNV Plasmid Transcription and Transfection

MNV plasmids were linearised with NotI and phenol/chloroform extracted before being used for in vitro transcription using the HiScribe^™ T7 ARCA mRNA Kit (NEB), following the manufacturer’s instructions. RNA was purified and concentrated using the RNA Clean and Concentrator Kit (Zymo). RNA/DNA transfection was carried out as previously described (62 (link)). GFP fluorescence at 24 and 48 hours post transfection was analysed via the Incucyte S3 machine (Sartorius).

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Mills J.T., Minogue S.C., Snowden J.S., Arden W.K., Rowlands D.J., Stonehouse N.J., Wobus C.E, & Herod M.R. (2023). Amino acid substitutions in norovirus VP1 dictate cell tropism via an attachment process dependent on membrane mobility. bioRxiv.

Publication Preprint 2023

HiScribe™ T7 ARCA mRNA Kit RNA Clean and Concentrator Kit Incucyte S3 machine

Chloroform Fluorescence Mrna Phenol Plasmids Transcription Transfection

Corresponding Organization : University of Leeds

Other organizations : University of Michigan–Ann Arbor

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Variable analysis

independent variables

Linearisation of MNV plasmids with NotI
In vitro transcription using the HiScribe™ T7 ARCA mRNA Kit (NEB)

dependent variables

GFP fluorescence at 24 and 48 hours post transfection

control variables

Manufacturer's instructions for the HiScribe™ T7 ARCA mRNA Kit (NEB) were followed
RNA was purified and concentrated using the RNA Clean and Concentrator Kit (Zymo)

controls

Positive control: None specified
Negative control: None specified

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