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Summary: Intratumor and gut mycobiome is linked to pancreatic ductal adenocarcinoma (PDAC) tumorigenesis; however, an optimal approach to culture and transplant fungus into mouse for in vivo studies is missing. This protocol describes culture steps of Alternaria alternata and Malassezia globosa and their subsequent transplantation into a PDAC mouse model via oral gavage. The utilization of the fungal culture method will allow for consistent growth and expansion of specific fungal species for downstream processing.For complete details on the use and execution of this protocol, please refer to Alam et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.