Journal: Fungal genetics reports
Article Title: A mus-51 RIP allele for transformation of Neurospora crassa
Figure Lengend Snippet: PCR analysis confirms that resistance to cyclosporin A is due to replacement of the csr-1 + allele with csr-1 Δ ∷hph . A) A diagram of the csr-1 + locus (top) relative to the csr-1 Δ ∷hph vector (bottom). Oligonucleotides P583 and P584 amplify a 3719 bp product from the csr-1 locus when it contains the csr-1 + allele and a 3500 bp PCR product from the locus when it contains the csr-1 Δ ∷hph allele. The restriction endonuclease Not I can be used to distinguish between these two PCR products of similar length. While Not I will digest a csr-1 Δ ∷hph PCR product into two fragments of 2473 and 1027 bp, it should not digest a PCR product from csr-1 + . B) Eleven transformants from the csr-1 + gene deletion assay were analyzed by PCR. The four host strains (host), a cyclosporin A-resistant transformant (CART) from each host, and a cyclosporin A-susceptible transformant (CAST) from each host (if available) were analyzed. The csr-1 locus in each strain was PCR amplified with oligonucleotides P583 and P584, digested with Not I, and analyzed by gel electrophoresis. Only CART strains (lanes 2, 5, 8, 11) and the positive control P8-65 (lane 12) produced PCR products with the expected pattern for the csr-1 Δ ∷hph allele. The transformants were not purified by homokaryon isolation, thus csr-1 + and csr-1 Δ ∷hph specific patterns in some lanes is expected ( e.g ., lanes 2, 8, and 11). The 5.0 and 1.5 kb positions, according to the included DNA ladder, are indicated to the left of the gel image. Lane information: kb) DNA ladder; 1) RZS27.4, host; 2) RZS27.4, CART; 3) RZS27.4, CAST; 4) RZS27.6, host; 5) RZS27.6, CART; 6) RZS27.6, CAST; 7) RZS27.10, host; 8) RZS27.10, CART; 9) RZS27.10, CAST; 10) RZS27.18, host; 11) RZS27.18, CART; 12) P8-65, csr-1 Δ ∷hph control; and 13) F2-26, csr-1 + control.
Article Snippet: The PCR product was purified with an IBI scientific Gel/PCR DNA Fragment Extraction Kit and 500 ng were electroporated into conidia as previously described ( ) with selection for hygromycin resistance.
Techniques: Polymerase Chain Reaction, Plasmid Preparation, DNA Deletion Assay, Amplification, Nucleic Acid Electrophoresis, Positive Control, Produced, Purification, Isolation