The NMD reporter plasmids have been previously described (Kolakada et al., 2024 (link)). For individual fluorescent and luminescent reporters containing Gly, Val, Ala, and Tyr amino acids before the PTC, oligos containing these sequences (Integrated DNA Technologies) and matching the EcoRI (NEB, R3101S) and XhoI (NEB, R0146S) restriction sites were synthesized. These oligos were annealed and ligated using the Quick Ligation Kit (NEB, M2200L) into EJC-independent and EJC-enhanced fluorescent and luminescent backbones digested with EcoRI and XhoI. To make the NMD– reporters for each sequence, each EJC-independent reporter was digested with EcoRI and MfeI (NEB, R3589S), the sticky ends were filled in using Klenow Fragment (NEB, M0212S) and the blunt ends were ligated together using the Quick Ligation Kit (NEB), removing the GFP 3′ UTR. The oligos synthesized for each sequence tested are as follows.
Protocol detail
Constructing NMD Reporter Plasmids
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Corresponding Organization : University of Colorado Anschutz Medical Campus
Other organizations : Engelhardt Institute of Molecular Biology
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Variable analysis
independent variables
- Amino acid sequences (Gly, Val, Ala, Tyr) before the PTC
- Fluorescent and luminescent reporter expression
- EcoRI and XhoI restriction sites used for cloning
- EJC-independent and EJC-enhanced reporter backbones
- Removal of the GFP 3' UTR from the EJC-independent reporters to create the NMD- reporters
- Positive control: EJC-independent and EJC-enhanced fluorescent and luminescent reporters
- Negative control: Not explicitly mentioned
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