Isolation of genomic DNA
Mammalian high molecular weight DNA can be prepared from a variety of sources. The choice is dictated by the availability of materials and the ease of preparation. Tissue culture cells are particularly convenient to work with. Isolation of nuclei prior to extraction of DNA is now done much more rarely than in the past, because, in most cases, mitochondrial DNA does not cause problems. The presence of small amounts of RNA does not inhibit restriction enzymes later on. Ethanol-precipitable material other than nucleic acids does not usually have a negative effect on further manipulations except that it may cause difficulties in dissolving high-molecular-weight DNA. If high-molecular-weight DNA is isolated for construction of genomic lambda libraries or genomic Southern blots the DNA should have a minimal size of 100-200 lb. For cosmid libraries larger DNA is needed. The protocol described here yields DNA of good quality with a standardized procedure designed to minimize the number of steps and manipulations. This is especially convenient if several samples are prepared in parallel. The size of the DNA can be ascertained in 0.3% agarose gels run at a low-voltage gradient. For a better view of the size distribution of the DNA, pulsed-field gradient gel electrophoresis can be used, but in practice DNA preparations that pass the standard gel test are satisfactory.
- OSTI ID:
- 6659839
- Journal Information:
- Methods Enzymol.; (United States), Vol. 152
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA
PURIFICATION
BIOCHEMISTRY
BLOOD CELLS
CELL CULTURES
ELECTROPHORESIS
KIDNEYS
LIVER
MAN
SPLEEN
ANIMALS
BIOLOGICAL MATERIALS
BLOOD
BODY
BODY FLUIDS
CHEMISTRY
DIGESTIVE SYSTEM
GLANDS
MAMMALS
MATERIALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PRIMATES
VERTEBRATES
550200* - Biochemistry