Southern Blotting: A Valuable Molecular Technique to Verify Gene Replacement in Ustilago maydis.

One of the most often applied procedures in the quest for gene function analysis in Ustilago maydis is to replace the target gene with an antibiotic resistance cassette by double homologous recombination with a linear gene replacement construct. Unfortunately, many unwanted events can also happen, which would go unnoticed if relying on PCR to verify the resulting strains. Therefore, it is recommended to verify the proper gene replacement using the easily doable and highly informative Southern blotting technique. This chapter provides a detailed and robust protocol for the verification of proper gene replacement in U. maydis mutants by non-radioactive Southern blotting.