Plasmid pUC19-PFBV contains the full-length PFBV genome (NC_005286) downstream of a T7 promoter and was synthesized by GenScript (Piscataway, NJ). Plasmid pUC19-PEMV2 contains the full-length PEMV2 RNA2 genome downstream of a T7 promoter (26 (link)). PFBV and PEMV2 mutants were generated by QuikChange one-step site-directed mutagenesis (28 (link)) using custom oligonucleotide primers (Integrated DNA Technologies) (Supplemental Table S1 ). Resultant PCR products were subjected to DpnI digestion and transformed into chemically competent DH5α E. coli cells. Mutations were confirmed by sequencing (Eurofins Genomics). The JIN-PTE-WT fragment (D86123; positions 3738–3880) and JIN-PTEmPK fragment were purchased as gBlocks Gene Fragments from Integrated DNA Technologies.
Protocol detail
Generating PFBV and PEMV2 Mutants
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Cells
Digestion
E coli
Gene
Genome
Mutations
Oligonucleotide primers
Plasmid
Site directed mutagenesis
Corresponding Organization : University of Maryland, College Park
Other organizations : Frederick National Laboratory for Cancer Research, National Cancer Institute, Center for Cancer Research
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Variable analysis
independent variables
- Mutations in PFBV and PEMV2 genomes
- Resultant PCR products
- T7 promoter used to drive the full-length PFBV and PEMV2 RNA2 genomes
- DH5α E. coli cells used for transformation
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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