Oliver Smithies:[00:00:00] Here we go on book little y starting June 2nd, 1980 and going through to August of the same year. It’s continuing the same technical problems of getting different clones or fragments. Alu subclones from the pseudogene. Appears straightforward matters Tuesday, June 30th, still thinking about Stockmayer equation with a little parenthesis there. Stockmayer for 2.0 parenthesis Jake. Jake was Fred Blattner’s son. [00:01:00] And Fred wrote a program to calculate the Stockmayer concentration. Simple computer program. And he called that program Jake. And so we would use this little computer program to calculate what the concentrations of vector and fragment should be to get optimum ligation. So here is an example of doing that again Tuesday, June 3rd, page three. But in this case [self-cloning?] will be no problem, so can work at one-tenth of the Stockmayer concentration. The following page continues with the result. Some (inaudible) grew rather poorly so was abandoned. [00:02:00] Suspiciously slow growth. Although their ligation was accessible. Start again with a different batch of medium. But still no good on the following page. So begin again with the transformation on Friday, June 6th and continuing. Of which a variety of colonies were chosen. Number 22 on page 13 was chosen as — or 3, 4, 22, and 27 were chosen. Sorry. Three, 4, and 22 were chosen as positives and 27 as a negative [00:03:00] [bacterium?] clone. Trying to clone these. Subclone these fragments. Note on page 19. This is my first recollection of the mentioning of John Devereux. He grew up number 4 and it checked out OK. That’s not particularly important. I just note that John Devereux is commented on. He was my technician for a number of years and together was responsible for publication of a whole variety of computer programs for analyzing DNA sequence. In fact of all the references [00:04:00] I have, the one I have with him on computer programs for use in analyzing DNA sequences of which he’s the first author and I’m the last has the most citations. I think it’s up in the 17,000 region at the moment. John was not terribly good at computer programs and not terribly good at DNA but he recruited a first-class computer scientist and was able to communicate with the computer scientist and with me on the DNA so I would suggest what they might write a program for, and John [00:05:00] could communicate with the programmer. And together we produced a whole set of fairly simple programs which were published in various forms as the Genetics Computer Group, GCG, programs from the University of Wisconsin. And the name of the computer person was — the name of the computer program was Paul Haeberli. And the paper eventually was called “A Comprehensive Set of Sequence Analysis Programs for the VAX Computer” published in Nucleic Acids Research in 1984. So this is the first mention that I have here of John. [00:06:00] So John Devereux. So he was in the lab in 1980. That paper was published in ’84. So he was with me for quite a time.
More work on plasmid (inaudible) 30.5 to get probes on page 21, July 1st, Tuesday. More probes, etc. in the next pages, and nothing very remarkable. Probes continue July 7th. Conclusion. Preps look OK. But query doubt if pBR322 [in this in this?] is real, etc. But not important anyway. So probes were being made of various types. Ligations and Stockmayer July 7th, Monday. [00:07:00] Page 35. More probes. More minis being [processed?]. Nothing particularly remarkable. Just continuing in the same way. Typical example of what was happening was page 44. The conclusion was that A — that was one particular clone — looks like pBR322. And B with a big checkmark looks correct. So clone. And following page it says now that D looks like B, exclamation mark, exclamation mark. That C looks correct and [00:08:00] comment on what they might be in making the probes. And is continuing rather screwy gel on page 53 very much the poor gel but the bands are quite clear. Bulk growths these A, B, C, D clones. Number 60, number 10, number 23, and number 27 on July 15th, Tuesday, page 55.
Oh. Getting back to some work with nuclei for DNA [swan?] digestion. Abe Worcel’s comment on how to do that type of experiment. [00:09:00] And his protocol on page 58. Cells from Natalie Borenstein. [TLC-L-19?] Wednesday, July 23rd. I don’t know whether we’ve talked much about Natalie but she was a grand technician who worked with me for a number of years and later with Bill Dove who when I went with my wife, with Nobuyo, to [00:10:00] North Carolina, Natalie Borenstein went to work with Bill Dove. And he always said that he owed two things to me. One was Natalie Borenstein and the other was that I gave him the name of my physician who was a top-class carer of people. But anyway there’s mention of Natalie on this page in looking at cells. Her mammalian cell K562, one type of cell, and [TLC-L-19?], another. Trying to make nuclei but abandoned the experiment on page 63 and set up again in the following days. [00:11:00] Isolating nuclei. Looking at DNA obtained on number 1 on page 69, Monday, July 28th. Concentration 23 micrograms per ml. The genomic DNA needs about 30 micrograms in about 100 microliters per slot. That was the type of Southern blot used at that time. And on the following page, 73, is an experiment with EcoRI digestions [00:12:00] of different lengths of time on this type of DNA. One of the samples being deliberately contaminated with lambda bacteriophage to see that the digest was going OK. As commented that the conclusion is the digests all look OK. And lambda bacteriophage is approximately complete. Meaning that the digestion had worked by 10 minutes given complete digestion. So that’s typical experiment of that type. Page 73. [00:13:00] And now the gels themselves being tested for transfer on page 75 and 74. And continuing. More gels on page 77. These different degrees of digestion, 79, 81 more repeats, just different gels.
Back to making nuclei again on Thursday, July 31st, page 83. And my concentration of nuclei obtained from K562 about [00:14:00] 10 to the 9 nuclei per ml. But work on improving the quality of the nuclei. The quantity is good. Some thoughts on the results on page 85, nothing particularly striking (inaudible) hybridization Saturday, August 2nd, etc. Just continuing with the same general type of work. [00:15:00] And typical comment on page 99. Run the experiment anyway. And underlined next time phenol after the restriction enzymes. Alu repeated Wednesday, August 6th, page 101. Nuclei again on following page. Various tests of trying to make good nuclei over the following pages. Different digests from earlier pages were now being treated with S1 nuclease on page 109. [00:16:00] y97 Alu Rsa Alu, etc. and various different things.
And only the pre, the one called pre, showed any hybridization. So that indicated problems. Repeating the Alu test. The only sure positive fully controlled result is with Alu, which should give a shorter piece than the [00:17:00] starting probe. Repeat of the Alu test Saturday, August 9th, page 111. Going repeating the work again. With S1 nuclease tested. Monday, August 11th, page 113. The result. y89 pre was the only one positive on previous experiment. And y111 Alu shows nothing overnight. Back on film. And so first conclusion is repeat these experiments with a single copy probe. [00:18:00] The pre is positive. And the samples having pre in them are positive but nothing else on page 113. Continuing in the same vein, 119, repeat of the S1 check with larger volumes. Nuclei again on the following page. S1 nuclease check continued page 125. With a comment. The digest looks good but still nothing much. [00:19:00] Most of the counts are at the dye front. That’s all that one could see in the particular experiment. So it wasn’t working.
And now another experiment with pBR322 clone 2.3 with a result saying that most of the counts are at the origin. Technical problems. Technical problems. [00:20:00] One thirty-five, still having difficulties. Wednesday, August 20th, 137. S1 nuclease conditions with very heavily outlined result. Unambiguous result. S1 nuclease cuts off these labels. Try therefore and see [p?], etc., etc., and repeat 30.5. So conclusion that it was back to stage one or stage [w?], exclamation mark, exclamation mark. [00:21:00] No immediate answers. Thinking about reannealing. C0t curves, which were very popular about that time. C0t curves, that was reannealing. And the time it takes for fragments to find each other or single-stranded material to find its partner and reanneal. The more repetitive a fragment is, the more likely it is to find and reanneal to its complementary or its opposite strand. More nuclei experiments on page 151, [00:22:00] and it looks like that is the end of this book, ending on still trying to make nuclei, book y.