Oliver Smithies:[00:00:00] Here we go with Book p, beginning April 6, 1977, and ending in July of the same year. And, we’re still talking about the colonies on filter paper, using chi1776 in this case. Electrophoretic transfer considered, and spreading on paper, re-spreading on paper later, and other filter paper with stained colonies on it. This is 1776; a digression really. [00:01:00] Getting sidetracked by 1776. There is a colony — there is a plasmid, p9 — pCR1, plasmids CR1, which apparently, I’m asking, has no blunt end restriction enzyme is available that cuts pCR1, a telephone message from Helen Armstrong; I must have asked from [Helinski’s?] lab. So here is pCR1, rough tests available. Digest it with [00:02:00] SmaI, EcoRI, [BamI?] control, and although the gel was [patently?] overloaded, EcoRI made a single cut, as expected. The control is about 90% supercircles. Bam didn’t cut; Sma didn’t cut. Not expected, but the enzyme is uncertain. So the only thing that clearly cut was EcoRI. Going with lambda, with chi1776 again, and repeat of SmaI on pCR1, with a new [00:03:00] preparation of SmaI, and there, the comment that SmaI (inaudible) as expected. And RI cuts more when in excess; Bam does not, so there are some contaminating enzymes presumably, and the SmaI.
And there are the maps on page 12.
And Leslie’s gels were being considered on April 14th. Incredible gels, very beautiful. There are markers in HaeIII, and HaeIII, extremely pretty gels on page 14. [00:04:00] And measuring the sizes in that way. The marker had about — I don’t know; these gels are just absolutely glorious. They look much better than the gels I was using. So these are polyacrylamide gels, almost certainly. With many, many bands. [00:05:00]
Another thought on how to put tails on things, on page 17.
A pCR1 digest on page 20 and 21, with EcoRI, and SalI, and the combined digest. “Cuts as expected, [00:06:00] use RI over 50, and use SalI undiluted.”
cDNA cloning with pCR1 on page 23, which was lost, so repeat. Repeat of this experiment, which Harvey had never had the loss problem before, so just try a straight repeat. [00:07:00] dT trials, and dA trial on page 27, but with an X right through everything. Continuing with PCR on April 21st, struggling. I’m happy with the p27 experiment, [00:08:00] which is the one with the big X through it. So it must be on page 26, we have to look on page 26, which is — terminal transferase with cobalt. PLTT number 3898-1, with dTTP, dATP, so giving page 27 dT; page 27 dA, these are the two products, dA and dT. And they’re being analyzed on page 31, and (inaudible) — come out fine. It’s saying that “Products are physically intact and looking good. Yield’s fine. dA, dT, [00:09:00] dA, dT [after?] terminal transferase.”
So cloning cDNA being considered on April 22nd. So, 15 microcuries total of dNTP is used, and the reverse transcriptase from Jeff Ross, and 3.9μL of globin mRNA, 2μg on page 33. So this was really good, [00:10:00] sensible work, using mRNA and reverse transcriptase from Jeff Ross to make cDNA clone. And to run down a column of SE [sepharose?], having washed with EDTA and treated with RNase. (inaudible) a column of SE sepharose on the top, and G-100 below to elute the cDNA, [00:11:00] with a comment on the left that the bulk of the radioactivity is still in the column, even though the fractions came out, and pools, fractions 6, 7, and 8 were pooled and precipitated with ethanol, representing about a 6% conversion of the mRNA to cDNA, and then into a Beckman poly-[alima?] tube, and to minus 20.
So that is considered the pages, page 33 is headed, well has a note in [00:12:00] a circle “clone,” and page 35 has “cloning again,” and those are tied to the whole page on the top saying “cloning,” so this is the cloning of the cDNA that is being carried out successfully I think in the end.
pCR1 cDNA continued. dTT tails, terminal transferase. And a comment [00:13:00] on page 36, “[Head will?] transform with all of the p37 mix, and with p29 dA,” so this is taking various preparations being used. Read this, written all caps. And the end, ultracentrifuge run, precipitate from the ultracentrifuge run, added sodium hydroxide and then, precipitated the material, [00:14:00] and recovered about 8900 counts per minute, whereas unrecovered was 96,000, 93,000 counts per minute, about 10%.
Repeat going on with the cloning on page 41, April 26th, repeat of the cDNA step to avoid losses, starting with 15 microcuries of, x3 of the dNTPs; the dNTPs being [00:15:00] dNTP mix. And let’s do that again.
15 microcuries x3 of the dNTPs. And, specific activity is 187.5 curies/mole with dNTP. So the last of the mRNA is being added in this experiment, much less than 3.9μL of globin [00:16:00] mRNA, about 1μL used for labeling with reverse transcriptase, etc. And eventually, it precipitated, after running through a column, SP40 EtOH cDNA, total 126,000 counts/minute of the cDNA.
And continuing the cloning on April 27th with terminal transferase, and partly my writing; partly someone else’s writing. Probably Harvey’s writing. [00:17:00]
On page 45, April 27th, Wednesday, “Closing the circle.” Model test in dA and dT. The aim is to anneal dA tails and dT tails, RI cut, pCR1, re-cut with SalI and then ligase. So it’s a complicated test, to see if it works. And then also, considering the stock marker concentration.
This DNA is on page 24. This DNA is 20% of lambda, and has a stock marker concentration of 0.075μg/μL, i.e. the present concentration, using the Stockmeyer equation. [00:18:00]
So slowly annealing on page 47 and 48, starting at 50 degrees, and lowering the temperature slowly, so that by two hours, we’re down to about 37 degrees, with a comment that, while this is annealing, the dTT cDNA to presumably dA-tailed — let’s just say — [00:19:00] yeah. So this is dT cDNA dT with tails of dTs on the ends, and the material to which it’s being annealed is p29 dA. And p29 dA is, pCR1, as I expected.
And continuing on page [00:20:00] 49, “Need to establish chemically the success of the various reactions for closure, since the transformation is at low efficiency, and somewhat erratic. So therefore, make more dA and dT tail material.” And Rabbitt’s protocol, or Rabbitt’s paper, on bacterial cloning of plasmids carrying copies of rabbit globin mRNA here detailed, because that’s basically what we’re trying to do, with pCR1. Rabbitt’s [00:21:00] was using Mini-ColEI plasmid cut with EcoRI terminal transferase with dTTP, and then one piece with dTT, and one piece with dATP, and the annealing of the two, just as I’m trying to do in this work here.
And another annealing experiment on page 52 and 53, temperature changing from 50 down to 37 over a period of several hours. Conclusion that “Re-annealing [00:22:00] with the plasmid is OK, but not with tail; must start again and do all the tails with 32P.” Didn’t get the result we wanted.
So pCR1, a restart on May 5th, and continuing on the next three pages. The corrected recipe on May 6th, and a result on page 61, with the tails being now radioactive. And, trying to select and using material that had been labeled with dAs for 60 minutes. [00:23:00]
So dA, dT and pCR1 again on page 63, with an error.
Closing the circle again, clone, on page 65. P65, annealed pCR1 globin, so it’s p43 — we’re in Book p — p43 dT, cDNA dT was added to dA, and pCR1, following Rabbitt’s procedure to give an annealed pCR1 globin, and Ed will transfect this material. And, with a comment that, “Using 20μL, [00:24:00] 3 putatives were obtained at about 2% efficiency of control pCR1, so that the frequencing of getting a colony was much reduced, but looked like there were colonies there.”
Physical closure being talked about on the next page, and conclusions, “This new material is poor; probably branched compared to the previous lots. Since it renatures as a polymer, but it’s worth trying the enzyme method on it.”
Alkaline tests on page 71, [00:25:00] material that had been treated with 0.4M sodium hydroxide, with a conclusion that the p63, page 63, page 63 from Book b, “dA is severely damaged, and can’t manage this long a time, but the material on page 49 is OK to continue with, as is page 63 material.” So, looking for which is the best product, after alkaline. Annealing again, with enzyme. [00:26:00] And the enzyme being SalI.
With some [reprint?] again from [Science?], a paper by Armstrong in Helinski’s lab, on the gene cloning, and containment properties of plasmid ColEI, and it’s derivatives. Remind me what else is going on, so here I am testing the results. Conclusions of [00:27:00] the annealing material, the assay works fine on page 75; the assay worked fine. It looks as if p63 is good stuff, and gives the expected product. Set up a large amount, and go on to ligase. So, here is ligation on Thursday, of SalI, pCR1. [To add?], for transformation.
More ligations in page 81, so continuing, we get ligation at 37 degrees with ligase at 5x the concentration. This is a test of Sal ligation [00:28:00] and of Sal inactivation, continuing these different ways of trying to get to clone cDNA. “Again, no ligation” is the conclusion on page 87, ”So ?does this Sal cut off the label p32?”
pCR1 EcoRI ligation. “[And guess?] the SalI enzyme is poor because the overlap is less good. Reverse the enzymes, and use SalI for tailing with EcoRI for closure, and test RI closure,” So that’s pCR1 with EcoRI [00:29:00] ligation. The conclusion is that “p89 ligase, or heat ligase in the cold works well, see page 101,” which is several pages further down. But we’ll sneak a look. The ligation is working, it says there, but we’ll get to that in due course.
These are different permutations of the possible things that can be done. Ligation at 37, or ligation, and then 90 minutes, etc., etc., heat and ligase, so on, ligation on page 93, still more tests to try to get a product that will survive. [00:30:00]
And then on page 99, Friday, May 29th, tests of dAT, dA, and dT annealed without cutting, (inaudible) given to (inaudible) product. Page 101, the catch-up gel with older samples. This is a result that was said, “Look at that.” And these gels are not my gels; almost certainly they look like polyacrylamide gels. Although, they are my gels. But it must — it’s a very good gel. And it’s Helling’s x4 0.7%, [00:31:00] it’s a high-resolution gel, very pleasing gel, with p63 dA, that’s page 63, Book p, dA and dT, and a new product, and the ligase cold, and the heat ligase, etc., etc. And the conclusion is, “The ligation is working with and without heat after RI; it’s not critical, nor is buffer. Stick with the page 83 material, or recipe. Sent the product to Ed, p101, anneal.”
The interpretations of the gel is given on page 100. “pCR1 closed [00:32:00] monomer, open monomer, open dimer, closed dimer, open trimer, closed trimer,” etc., trying to interpret, probably correctly, what they were. “And given to Ed.”
Page 103, Saturday, May 21st, “Endure the Sal ligation, in view of the current success with RI ligation, and stock of dA and dT tailed, RI-cut plasmids can go back to Sal test,” and test that — however I found that I’ve done this experiment already. And better stick with EcoRI for the second cut.
New pCR1, Sal/EcoRI series done. [00:33:00] Conclusion, “All is well. The recovery’s fine. And you can see post-lambda [XO?] is more concentrated,” etc.
Start of (inaudible) transferase on the Sunday, the 22nd, page 109. And continuing from that page, and cDNA on May 23rd, with, after the cDNA mix with dNTPs, and globin mRNA, 2μgs. “OK to proceed, but in future, this step might best be done with dialysis, [Eave’s?] solution, use about 10-20%.” [00:34:00]
Again column on my next page, SE Sephadex on the top, followed by Sephadex G-100, and obtained a pool of product. This is material put on the column was not clear.
This is cDNA made on the preceding page, page 113, cDNA made on this page, and then cleaned up and (inaudible), with a possible error. [00:35:00]
And on page 117, recover the cDNA, and the second dT tail, with a conclusion that, “Once again, dialysis will be better.”
Continuing to have problems, page 119, (inaudible) [degrees?]. Cloning test, a rough test on page 121. Did not work. [00:36:00] Restart with dialysis only, this time, page 123. The conclusion that, “The Sal cut is adequate. The recovery is greater than 50%. Continue and re-titrate after terminal transferase.” Lambda exo was used to cut the [00:37:00] —
I guess we’re going to start again on page 123 of Book p. We’ll restart with dialysis only, where pCR1 DNA was treated with SalI, and then after recovering the material, it was treated with lambda XO to make single-stranded ends, and kept on ice. [00:38:00]
Continuing to struggle, terminal transferase on page 127 again. Sal-dT, so it’s Sal cut with dT tails, the idea, cDNA again on page 131, and pCR1 Sal-dT is available, that’s been cut with Sal and dT ends added.
Terminal transferase, #2 experiment, June 1st. So the conclusion on 135, “p133 material dT, cDNA dT, has just about, and only [00:39:00] just, survived.
So annealing test on June 2nd. This all seems to be rather negative. So, the experiment is starting with [00:40:00] material, pCR1 Sal digested with dT, cDNA dTT, available in terminal transferase buffer, and pCR1 Sal-dA available also in terminal transferase buffer, and then ligated them, or annealed them, anyway, not ligated, annealed these. And then, ligase. [00:41:00] [Ned?] will take the 20μL for tests on Saturday of this material, that’s the ligation product. Although the conclusions are, “It doesn’t look like a good annealing step, on the check on the gels.”
So, remake the globin ligation after a rest for going to the National Institute of Health, and flying. This was June 6th, the gap [00:42:00] to June 20th. Ed reports that the material from page 139, ligation was the best product we have, but he’s now run out of material, so we have to remake material. And, a globin ligation made again with 20μL, the last of post-EcoRI sample made on page 137. There’s a ligase there, and this product is called, “p143 pCR1 globin ligation. And Ed used this with great success,” is the conclusion. [00:43:00]
Page 145, “The globin cDNA probe,” is the heading. Globin mRNA is available at 0.5μg/μL, and Harvey’s protocol describes that. “Isophenol, and reverse transcriptase,” etc., etc., “sodium hydroxide, phenol extraction, and then on a column with [00:44:00] material eluting and basically fraction [seven?], or six and eight, pool of p145 globin probe, 3.2×10^6 counts per minute.” And so, etc., “Finally 2,660 counts per microliter product, of globin cDNA [pool?].
July 6th, after a week of cross-country flying. The available [gluten?], blocks for immunoglobulin plasmid. [00:45:00] [00:46:00]
A nice immunoglobulin cDNA, made on page 149. Terminal transferase on page 151.
And catch-up gel on page 153, of the material annealed, annealing with Sal cDNA dT, [00:47:00] and Sal dA annealed with ligase, etc. And with a conclusion that, “The ligation’s proceeding OK. Traces of the desired product probably present.” This is immunoglobulin plasmid on page 153.
Ending on the following page, with the last check on the p152 ligation, that RI, post-RI IgG plasmid, and pCR1 IgG ligation, undiluted. And, pCR1 marker. Conclusion: no significant change since [00:48:00] 12 hours. And that’s page 155, of Book p, and the end of this book.