A simplified mammalian DNA isolation procedure has been developed to minimize the number of manipulations required for each sample. This method avoids centrifugation steps and organic solvent extractions. The basic procedure involves three steps: lysis, isopropanol precipitation, and recovery of the precipitate. The lysis buffer contains components that allow restriction digestion of DNA without prior organic solvent extractions. The lysis buffer is added to the tissue or cells, and digestion is complete within several hours at 37°C (for cells) or 55°C (for tissues) with agitation. Isopropanol is then added to the lysate, and the samples are mixed until precipitation is complete. The DNA is recovered by lifting the precipitate and dispersing it in a prelabeled tube with TE buffer. The DNA is then dissolved by agitation at 37°C or 55°C. This method is applicable to cultured cells and tail biopsies. For cultured cells, the DNA isolation can be performed in the same well used for cell culture. For tail biopsies, a centrifugation step is required to remove hairs and tissue residue. The DNA obtained is of sufficient quality for Southern blot analysis using various restriction enzymes. The procedure has been used routinely in several mouse genetics laboratories due to its reduced manual labor. If problems occur, a phenol/chloroform extraction followed by reprecipitation can be used. This method provides higher DNA yields compared to traditional methods and is suitable for analyzing fragments up to 20 kb.A simplified mammalian DNA isolation procedure has been developed to minimize the number of manipulations required for each sample. This method avoids centrifugation steps and organic solvent extractions. The basic procedure involves three steps: lysis, isopropanol precipitation, and recovery of the precipitate. The lysis buffer contains components that allow restriction digestion of DNA without prior organic solvent extractions. The lysis buffer is added to the tissue or cells, and digestion is complete within several hours at 37°C (for cells) or 55°C (for tissues) with agitation. Isopropanol is then added to the lysate, and the samples are mixed until precipitation is complete. The DNA is recovered by lifting the precipitate and dispersing it in a prelabeled tube with TE buffer. The DNA is then dissolved by agitation at 37°C or 55°C. This method is applicable to cultured cells and tail biopsies. For cultured cells, the DNA isolation can be performed in the same well used for cell culture. For tail biopsies, a centrifugation step is required to remove hairs and tissue residue. The DNA obtained is of sufficient quality for Southern blot analysis using various restriction enzymes. The procedure has been used routinely in several mouse genetics laboratories due to its reduced manual labor. If problems occur, a phenol/chloroform extraction followed by reprecipitation can be used. This method provides higher DNA yields compared to traditional methods and is suitable for analyzing fragments up to 20 kb.