Oliver Smithies:[00:00:00] In 1962, it goes on to ’63, and there is some beginning at first of talking about DNA-associated proteins, and the methods of separations we had used, and again, with not any very clear reason for doing these experiments at this point. Still going on with preparations of nuclei again on page 9 and 11, and so on. [00:01:00] Other tests of homogenization, pH, et cetera. Homogenizing in different ways, but why I was doing these experiments at all is very unclear to me looking back. (laughter) I don’t remember why I did anything with histones at that point at my career.
But here I am testing glycerol concentrations on page 29, Monday November 12th, so still trying to get good nuclei, and this is going on for many pages, filtration tests, and [Philpot?] medium.
Thursday November 15th, Philpot 2 [00:02:00] made without glycerol; final pH 7.1, et cetera, with glycerol. But for what purpose is a mystery.
Continuing in page 47 and 49, now liver being homogenized without glycerol, and with glycerol, et cetera, and diagrams of where the nuclei where, page 55, unclumped mitochondria, slightly mitochondria, markedly clumped mitochondria, debris, pellet resuspended. But why? A mystery.
Other gradients on November 21st. It looks like this book is all, entirely related to preparing histones again. [00:03:00]
New fractionator tested on Tuesday, November 27th, trying to have some way of directing different fractions into different tubes, of different parts of a centrifuge tube. And continuing, page 77, it goes on and on and on.
Agar again on Friday, December 7th, “In view of recent difficulty, decided to retest the agar separation with knowledge that nuclear agglutination requires glycerol. Pages and [00:04:00] pages of this stuff.
Larger scale idea on Sunday, December 9th. Still doing the same sort of work. Liver perfused this time, page 97, page 99, going on with this, (inaudible), retesting the homogenization, 24 strokes with fine-clearance rubber pestle, largely nuclei on page 103. Tests of other homogenizers, 105. Very easy to go through this book. It’s just trying to make nuclei. Tested four livers, pre-perfused [00:05:00] on page 119, tested without perfusion on page 121. On page 139, with no change in motivation still.
January 7th, rechecks of different homogenate. And so on, we’re coming to the end of this book. And at the end of the book, the general [00:06:00] conclusions of the nuclei from tumor are much less stable, presumably due to enzymes from necrotic tissue. The density is therefore usually low, and only rapid cold work can we expect good nuclei when necrosis is present, pre-dissection, very important. But again, why I was doing this work in this book is not at all clear.