June 17, 1991 | Peter W.Laird, Alice Zijderveld, Koert Linders, Michael A.Rudnicki, Rudolf Jaenisch and Anton Berns
This article describes a simplified mammalian DNA isolation procedure that minimizes the number of manipulations required for each sample, making it more efficient for gene function studies in mice. Traditional methods often require centrifugation and organic solvent extractions, but this new method eliminates these steps. The basic procedure involves three main steps: lysis of the tissue or cells in a lysis buffer, isopropanol precipitation, and recovery of the DNA in TE buffer. The lysis buffer contains Tris.HCl, EDTA, SDS, NaCl, and Proteinase K, allowing for restriction digestion without prior extraction. The procedure is particularly effective for cultured cells, as it can be performed in the same well used for cell culture. For tail biopsies, a brief centrifugation step is needed to remove debris. The DNA obtained is of high quality, suitable for Southern blot analysis with various restriction enzymes, including those active in low salt conditions. The method has been successfully applied to analyze DNA fragments up to 20 kb. The procedure is now used routinely in several mouse genetics laboratories due to its reduced manual labor. However, issues may arise from large tissue samples, insufficient agitation, or incomplete DNA dissolution. If problems occur, a phenol/chloroform extraction followed by reprecipitation can be used as an alternative. This simplified method improves the efficiency and reliability of DNA isolation for genetic studies.This article describes a simplified mammalian DNA isolation procedure that minimizes the number of manipulations required for each sample, making it more efficient for gene function studies in mice. Traditional methods often require centrifugation and organic solvent extractions, but this new method eliminates these steps. The basic procedure involves three main steps: lysis of the tissue or cells in a lysis buffer, isopropanol precipitation, and recovery of the DNA in TE buffer. The lysis buffer contains Tris.HCl, EDTA, SDS, NaCl, and Proteinase K, allowing for restriction digestion without prior extraction. The procedure is particularly effective for cultured cells, as it can be performed in the same well used for cell culture. For tail biopsies, a brief centrifugation step is needed to remove debris. The DNA obtained is of high quality, suitable for Southern blot analysis with various restriction enzymes, including those active in low salt conditions. The method has been successfully applied to analyze DNA fragments up to 20 kb. The procedure is now used routinely in several mouse genetics laboratories due to its reduced manual labor. However, issues may arise from large tissue samples, insufficient agitation, or incomplete DNA dissolution. If problems occur, a phenol/chloroform extraction followed by reprecipitation can be used as an alternative. This simplified method improves the efficiency and reliability of DNA isolation for genetic studies.