Oliver Smithies:[00:00:00] This is the beginning Book f, September 11th, 1973. I’m still in Madison, goes through November, so it’s a fairly brief period of time, and many pages of notes, looking for still the various elements needed to test this idea of branch DNA. And at the same time, crosslinking experiments going on. More bacterial crosses on page 11, September 14th. [00:01:00] And the 15th, 16th, 17th, 450-lambda plasmids again. New solutions for lysis, lysozyme and magnesium sulfate titrations.
Thinking about it, these were probably some of these mutants were — some of the bacterial [00:02:00] strains had lambda, but didn’t lyse, and so if you induced the phage, you would get phage DNA in the cell, but not lysis, which enabled the preparation of the lambda bacteriophage DNA, by lysing the cells well after purification, and the same thing for lambda dv.
Trying to induce mutations with ultraviolet, [00:03:00] or induce a cross, Wednesday, September 26th.
Now getting back to labeling again, labeled DNA, etc., Thursday, September 27th, 31, lambda dv, 0, 0.8 and 1.5. The numbers refer to previous notes. That’s to say the 0, 0.8, and 1.5. [00:04:00] The comments on page 47, “Lambda dv 0, spread at 1X, very dirty, DNA not in film. Lambda dv 0.8, as much DNA in the film, but dirty. Looks like E. coli.” But obviously, there’s a lower concentration, but it has nice DNA, [field?] with two circles, and some E. coli, hopefully we see these images. “Lambda dv 1.5 has much DNA, some circles, and [00:05:00] more dilute concentration field with two circles, and some E. coli. Hoping to see some images from the electron microscope, that we get through October, beginning of October.
For example, here we are, reports on the electron microscope, but no images on page 65, October 9th, F450(lambda with nuclease) [00:06:00] (inaudible). [D?] reports that, “The 0.8 at 450-lambda was not any good, as far as circles are concerned.” Rough try deletion from our F55 phages, and our method is to UV the phage for about 40 seconds, and very lightly, since UV-RB, with the host, etc., and trying to get different mutants, or different recombinants. Better try for a deletion here. And so it goes on, [00:07:00] pages and pages of bacterial genetics.
And here and then a chemistry interlude. Thursday, October 18th, cesium chloride protocol, how to get the products that one hopes to get, with a diagram on the opposite page, suggestion that different tubes had different products in them, with a gradient after putting the DNA on a density gradient, centrifugation, cesium chloride experiment. [00:08:00]
This is rather interesting, because it’s an insert on several pages of [R Helling?], April 1973, agarose gel electrophoresis of linear duplex DNA, and that is, what we can recall, the Helling’s buffer under 4X and 3X Helling’s were used many times in experiments later with DNA constructs. So this is the, from Helling himself, presumably, at least at the top it says “R Helling, [00:09:00] April ’73.” And ideas on the same page, “Then,” for example, “you use R1,” etc., “restriction enzymes for chromatin solubilization, and try DNA electrophoresis for making C50, lambda, and lambda DV with a tube containing 0.3% agarose, and [cellophane?] and electrophoresing through it to make a gel.”
And here is probably one of my first DNA electrophoresis experiments on Friday, October 26th, on page 82 and 83. [00:10:00] The gels contain DNA, all right, but the electrophoresis is pretty bad by my current knowledge of what can be done, but it’s beginning of electrophoresis of DNA, which later became very routine. And it includes looking at 450 lambda, R12, lambda dv, and lambda DNA [OS?] etc., lambda DNA from Fred [Blatner?]. [00:11:00] The gel showed that really that the lambda was readily visible with a number of bands. It must have been digested, I presume, not quite sure. But the [exhibiting/beginning?] of electrophoresis of (inaudible) sample.
Again, on the following pages, shorter timeline to dv now, showing something. R12, and lambda, and seeing the differences in migration. [00:12:00] I’m beginning to get somewhat better electrophoresis, as the time goes on. But still, very much asymmetric bands. And, trying to recover DNA from the gels [00:13:00] by cutting up the gel, freezing it, and recovering the liquid. An example of this on page 90, Friday, November 2nd.
Notes from someone else’s note, not my note, of putting dv into E. coli. So lambda dv again, Tuesday, November 6th, [see/FC?] f99, now looking at the DNAs [00:14:00] directly, rather than having to wait for electron microscopy. The gels are looking a little bit better. And starting to describe the buffer, as Hennings — it really should be Helling — Hennings 1x buffer at 20V/cm, and the electrophoresis is getting a little better. And now, this notation is clear, lambda dv DNA on page 101, in a buffer, Hellings Hx1, 20V/cm, and lambda dv on the gel. [00:15:00]
With comments on the results from preparations studied on the gel, but on page f101, 101p is quite clean, only one small circle, and 12 or so squares. F101, F was very dirty, but four or five small circles among the debris, and F101S, quite dirty, no DNA. Thus, results are approximately as expected with gel [00:16:00] run on the same page as 102 and 103.
Back to lambda dv on page 105, the gel’s again, 1% agar, Hx1, and preparative lambda dv gels on page 109. [00:17:00] And so, I’m beginning to digest with restriction enzymes that I’ve noticed at this point, F11 digest from Pat, in [Fred Blatner’s?] lab was, etc., etc., 50μL of restriction enzyme, and I imagine that was EcoRI at that time; not many restriction enzymes had been isolated. That was probably what we just called it, restriction enzyme. Still looking at digests now.
With one [00:18:00] gel on page 113, where the markers now are digest of the phage DNA, of lambda DNA, clearly, and quite good run, but nothing very much commented on. This is the bottom right-hand gel, the right sample. [00:19:00]
I’m trying to combine [dissection?] of lytic phages, or other type of bacteriophages, in a gel, DNA transfection — we’re using a gel strip — an idea on page 115. [00:20:00] Now looking at the electrophoresis on page 117, with the gels on page 116, the electrophoresis is very much better, but still having [Stan’s 1?] buffer, with 1% [C-chem?] gel. Not any real comment on how much the electrophoresis is getting better with time, but very obvious.
Some comments on the following page, 118, that the most likely conclusions are that the voltage per centimeter has a large effect on general migration, and the [00:21:00] temperature has no obvious effect, but increasing buffer probably has little effect at low volts per centimeter, but try the effect of gel concentration. And also, on page 119, try salt concentration. And the first mention of a Helling’s 4 buffer, 8×4, which later became our standard. A rough test of lambda DNA, etc., and [Fred Blatner’s?] digestion of it, 15 degrees at 10V/cm, and 2.5V/cm with [00:22:00] Helling’s x4. It looks like a pretty good gel. That’s on page 188. The left-hand gel is 15 degrees, 10V/cm, and the right-hand gel is 4x buffer, Helling’s 4x, at less than or equal to 2.5V/cm, and it’s a pretty nice electrophoresis. Both of them are quite good, but that’s the first mention of Helling’s 4 that I can see which as I say, later became standard.
More lambda dv powder on page 121. Exponential versus saturated cultures, that later became a very critical [00:23:00] matter in making the experiments work when we were doing gene targeting, but here I evidently had realized the importance of understanding the effects of exponential growth versus saturating stationary phase, with a comment on (inaudible), but cells are a smaller volume when stationary. Exponential, and stationary. The exponential, 1L of 9.6*10^7/mL, and 0.1L of stationary at 8*10^8/mL, so in effect, one was nearly 100 times more [00:24:00] cells in the stationary than the exponential.
Back to gels, with a changing concentration of DNA, at 2%, 1%, and 0.5%, “As expected, for the large molecules, must use the lowest practical gel concentration, the lowest V/cm, and ?the highest salt” which is what we eventually began to do. These are all Helling’s x1 on page 125.
Buffer concentration test on the following page 127, [00:25:00] then 1x, 2x, 4x, Helling’s. Result shows appreciable improvement at 4x; therefore the best conditions for the really high molecular weight DNA should be Helling’s x4 buffer, [C-chem?] 0.5%, 1V/cm, and run for at least 15 hours when cold. And later on, we changed that and we ran as hot as possible with high voltage, but nonetheless, it was Helling’s 4x buffer that became our standard. Our usual sort of progressive investigation of the variables to get the best result of the electrophoresis. Nice, systematic [00:26:00] work.
Large DNA tests on page 131, November 20th. And no particular comments, but the electrophoresis is quite nice to see. I’m beginning to think about radioactive material again. 14-C accounting. [00:27:00] How did I do that experiment?
Phenol tests on Wednesday, November 21st, trying to clean up the DNA with phenol extractions, with a page 137 note, “Don’t forget to try — this looks like a lambda dv with a replication half, about a third replicated, plus or minus ligand, to get (inaudible) for tetramer — [00:28:00] — plus monomer,”
Working on Thanksgiving day, preparing DNA from gel, Thursday, November 22nd. Henning’s, badly spelt, it should be Helling’s 20x stock and R1 buffer, so it is EcoRI that we’re using. Lambda dv and magnesium again, Friday, November 23rd, looking at the products on the gel. [00:29:00] And repeat of long-term lambda experiments at 2V/cm, Saturday, November 27th, with a comment that, need better control of voltage gradient, and on gel thickness in these experiments, because they’re very wavy [00:30:00] sample migration patterns. Ending, as usual, with a list of strains on page 149 and 148 of the bacterial strains. This must have been discipline that I’ve been taught by Fred or by [Bill Dove?], and that’s the end of Book f.