Preparation of high-molecular-weight DNA from Drosophila embryos.
Standard methods for extracting DNA from cells or organisms (e.g., phenol extraction and ethanol precipitation) produce fragments with an average size of 50-200 kb under optimal conditions. The shearing forces that are applied to DNA in solution during mechanical vortexing or mixing and pipetting produce frequent double-stranded breaks. To prepare high-molecular-weight (HMW) DNA, it is necessary to guard against such damaging forces by performing all extractions and manipulations on DNA that is embedded within a protective matrix. Preparation of HMW DNA from Drosophila embryos is described in detail here because, in our hands, it is the simplest and most reliable protocol and can be used for large- or small-scale preparations. The overall strategy is to purify nuclei, gently embed them in molten agarose, and then extract proteins and perform other enzymatic reactions by transferring the solidified agarose block into the appropriate solutions. Salts, soaps, and enzymes act on the DNA by diffusing through the agarose matrix, while the matrix protects the DNA from shearing forces.
Karpen
Gary H
GH
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004249:DNA Damage; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D005821:Genetic Techniques; D008970:Molecular Weight; D012685:Sepharose / Q000737:chemistry; D013314:Stress, Mechanical
D004247:DNA; D012685:Sepharose