An Efficient Colony PCR Method for Construct Verification in the Basidiomycete Ustilago maydis.

Rapid advances in AI-driven annotation and automation are increasing the demand for fast and reliable screening protocols for reverse genetic approaches. Traditional methods for genomic DNA extraction from the basidiomycete Ustilago maydis are often time-consuming and require expensive reagents and hazardous chemicals, making them unsuitable for large-scale mutant screening. Existing colony PCR protocols using crude lysates without DNA precipitation are fast but often fail in terms of reproducibility because cellular components can interfere with polymerase activity or DNA stability. These limitations can be addressed by adapting the yeast GC-prep protocol of Blount et al., based on glass-bead lysis and boiling in the presence of the metal-chelating resin Chelex-100, and combining it with the impurity-tolerant Phire Hot Start II DNA Polymerase. Here, we present a straightforward and reliable protocol for DNA isolation and storage in the screening of U. maydis mutants, enabling the reproducible amplification of 0.5-5 kb fragments directly from fresh colonies grown on agar plates.