This kit constitutes an all-in-one system for the rapid isolation of DNA from sputum samples. It allows for the isolation of bacterial or eukaryotic DNA from the sputum samples using a convenient spin column format. The kit includes all the reagents needed to process sputum DNA including a sputum liquifier to reduce viscosity and facilitate DNA isolation. The protocol can be completed in 30 minutes. Purified DNA is of the highest quality and free from inhibitors, and can be used in sensitive downstream applications including PCR and qPCR.