High prime dna labeling and detection starter kit 1

The putative mutants were analysed further by PCR. The primer pairs Qip-Up-3F/3R and Qip-Down-3F/3R were used to test for homologous recombination at the upstream and downstream flanking sequences. Primer pair Qip-F/R (Table 1) amplified a 1.1-kb fragment in the wild-type but not in the Qip deletion mutant. Additionally, genomic DNAs from the wild-type A8 strain and Qip deletion mutants were extracted by the method of CTAB. The hygromycin resistance gene’s copy numbers were analysed by genomic Southern blots using a digoxigenin labelling and detection system (High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Beijing, China) according to the standard protocol of the manufacturer.

The split-marker approach and PEG-mediated protoplast transformation [25 (link),26 (link)] were employed to construct Fgporin deletion mutants. Hygromycin-resistant transformants were identified using PCR with the specified primers (Supplementary Table S1) and Southern blotting. Southern blot hybridization was prepared using the High Prime DNA Labeling and Detection Starter kit I (Roche Diagnostics, Mannheim, Germany) to confirm single-copy integration. For complementary analysis, a recombinant pFL2 vector containing the full-length Fgporin with its promoter sequence was introduced into the protoplasts of the Δporin mutant; after geneticin (BBI Life Sciences Ltd., Beijing, China) screening on PDA plates, geneticin-resistant transformants were verified by PCR as the complemented strains. Similar approaches were used to generate FgUps1/2 and Fgmdm35 mutants.

Genomic DNA was extracted from leaf of non-transgenic and the putative T₁GmCnx1 transgenic plants (based on herbicide resistance, mentioned above) by the method described by Edwards et al.[22 (link)] with minor modifications. Plasmid DNA and genomic DNA were digested with restriction enzymes EcoR I and Hind Ⅲ, respectively, at 37°C overnight. Digested DNA was separated on 0.8% (w/v) agarose gel and blotted onto Hybond N⁺ nylon membrane. The membrane was hybridized with a DIG-labelled GmCnx1-specific probe (5’-CGAGTATCCCGTCGTTG-3’ and 5’-TTCCAGGTAGCCCAAAA-3’) at 68°C for 16 h. The hybridized membrane was washed and detected according to the protocol of High Prime DNA Labeling and Detection Starter Kit I (Roche, USA).