The Old Timey Maxiprep Protocol
One of my biggest fans got me a copy of The Eighth Day of Creation, which I’d been wanting to read for some time now. In the first chapter, check out this old-timey maxiprep (i.e. large-scale DNA isolation and purification) protocol, courtesy of Avery, MacLeod, and McCarty from their historic 1944 paper demonstrating that DNA was the carrier of genetic information in cells:
To get [DNA], they grew virulent Type III pneumococci at blood heat in twenty-gallon vats of broth made from beef hearts, spun out the bacilli in an iced centrifuge, suspended them in brine, and brought the “thick, creamy suspension of cells” quickly to a temperature hot enough to kill the cells and to inactivate “the intracellular enzyme known to destroy the transforming principle” [DNase]… They then washed the cooked pneumococci in three changes of brine to remove capsular sugar as well as whatever protein would come away, extracted the bacteria by shaking them for an hour in a solution of bile salt to break the cell walls (and then threw away the cell residue), and reprecipitated the extract with pure grain alcohol.
“The precipitate forms a fibrous mass which floats to the surface of the alcohol and can be removed directly by lifting it out with a spatula,” the paper said. This was now washed several times with chloroform to remove protein, and suspended yet again. A digestive enzyme was put in to eat away any remaining capsular sugar. Removal of protein was repeated, “until no further film of protein-chloroform gel is visible at the interface.” Pure grain alcohol was added again, “dropwise to the solution with constant stirring.” At a concentration where the alcohol nearly equalled the extract, “the active material separates out in the form of fibrous strands that wind themselves around the stirring rod. This precipitate is removed on the rod and washed….The yield of fibrous material obtained by this method varies from ten to twenty-five milligrams per seventy-five liters of culture.”
Wow, and I thought maxipreps take too much time now! Compare the above to a more modern DNA isolation protocol, courtesy of Black Knight.
