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Summary: The use of germ-free mice is integral to the understanding of host-gut microbiome relationships. Such models rely on faithful replication of the donor microbiome to establish causal effects of the gut microbiota on host pathophysiology. This protocol describes the preparation and transfer of donor microbiota, focusing on strict anaerobic processing methods and multiple instillations by gavage for optimal gut microbiota recovery.For complete details on the generation and use of this protocol, please refer to Choo and Rogers (2021).