A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources.

A rapid and simple procedure is described for obtaining CsCl-purified DNA from multiple small samples of cells or tissue. The DNA is recovered in a high-molecular-weight form (greater than or equal to 50 kb) that is readily cleaved with restriction enzymes. Sufficient quantities of DNA (10-50 micrograms) are recovered to allow multiple analyses by Southern blotting and most cloning procedures. The isolation procedure involves addition of intact cells or powders of frozen tissues directly to a simple lysis buffer containing detergent (sodium dodecyl sulfate or sodium sarcosinate) and high concentrations of EDTA. Ultra-high-speed centrifugation of CsCl gradients allows the isolation of DNA from 10 different samples in as little as 5 h. Applications are described for mammalian cells (HeLa cells), insect tissues (Drosophila melanogaster adults and pupa, Manduca sexta pupa, and Musca domestica pupa), higher plant tissues (Vicia faba leaves and meristems), algal cells (walled and wall-less Chlamydomonas reinhardi), yeast cells (Saccharomyces cerevisiae), and bacterial cells (Escherichia coli spheroplasts for preparation of both chromosomal and plasmid DNA). The procedure can be scaled up with larger sample sizes and longer centrifugation times to provide bulk quantities of DNA.

Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation.

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient.

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.