Generating Mutant CYVaV Plasmids

Plasmid pET17b-CYVaV, which contains the entire length of WT CYVaV genomic RNA (gRNA) complementary DNA (Accession number JX101610) downstream of a T7 promoter (33 (link)), was used as a template for polymerase chain reaction (PCR)-based site-directed mutagenesis. The desired mutations were introduced using custom-designed oligonucleotide primers (Integrated DNA Technologies) using Q5 high-fidelity DNA polymerase (New England Biolabs). The resulting PCR products were subjected to DpnI digestion followed by T4 DNA ligase before transformation into DH5α Escherichia coli cells. The presence of the desired mutations was confirmed through Sanger sequencing (Eurofins Genomics).

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Mikkelsen A.A., Gao F., Carino E., Bera S, & Simon A.E. (2023). -1 Programmed ribosomal frameshifting in Class 2 umbravirus-like RNAs uses multiple long-distance interactions to shift between active and inactive structures and destabilize the frameshift stimulating element. Nucleic Acids Research, 51(19), 10700-10718.

Publication 2023

oligonucleotide primers Q5 high-fidelity DNA polymerase Sanger sequencing

Cells Complementary dna Complementary rna Digestion Dna polymerase Escherichia coli Genomic Mutations Oligonucleotide primers Plasmid Polymerase chain reaction Site directed mutagenesis T4 dna ligase

Corresponding Organization :

Other organizations : University of Maryland, College Park

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Variable analysis

independent variables

The desired mutations introduced using custom-designed oligonucleotide primers

dependent variables

The presence of the desired mutations

control variables

Plasmid pET17b-CYVaV containing the entire length of WT CYVaV genomic RNA (gRNA) complementary DNA (Accession number JX101610) downstream of a T7 promoter
Q5 high-fidelity DNA polymerase (New England Biolabs) used for PCR-based site-directed mutagenesis
DpnI digestion and T4 DNA ligase treatment of the resulting PCR products
Transformation into DH5α Escherichia coli cells

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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