Oliver Smithies:

[00:00:00] Book kappa.  February 14th, 1985 going through July of the same year.  And I know from long history that this is the book in which we finally get to the proof that gene targeting is possible.  So here starts with getting ready for another packaging bulk digest of new preparation of Charon phage delta delta Xba.  Delta delta X.  Digestion is complete.  Start of sib selection.  Now this is important.  Because the procedure was aimed in the following way, that we would start with [00:01:00] Ron Gregg’s material.  Initially he had taken a pool of colonies.  And we’d established that we had within the pool recombinant bacteriophages.  So we knew that it was possible to target.  But we had not yet isolated the mammalian colonies which were positive.  So let me say that again.  We knew that targeting was achieved but it was not yet possible to isolate the mammalian cell in which that had been accomplished.  So sib selection is a method that had been used by many people in the past.  And that’s to say that you [00:02:00] start with a large number of individual colonies.  Purified colonies.  And you pool them.  Let’s say you put 1,000 individual colonies into a mixture.  And you determine that there’s a positive in that 1,000.  And then you take the 1,000 colonies or you take the colonies from which you made 1,000 and you plate them out and you mix them now in 10 pools of 100.  So the same 1,000 colonies are now distributed into 10 hundreds.  And you do your bulk assay and find out that maybe the [00:03:00] 400-through-500 batch contains the positive.  So you can throw away all the other 900.  Now you take the 100 colonies which were positive and regroup them into tens.  So you have 10 groups of 10.  And then you find out which of the 10 groups of 10 is positive and now you can look at each one individually, even though it might be quite hard work on the individual colony.  But you’ve sib-selected until you know that you have a small number of possible positives.  So here was the start of sib selection on the ligation of Ron Gregg’s preparation P31.5B.  [00:04:00] This was DNA from 98 colonies from an experiment with delta beta 117.  And that material was packaged in the usual way with freeze-thaw lysate and sonic extract.  And comment that this experiment was going to be with the whole of one of his pools there.  And it would require 100 microliters of the LB12 freeze-thaw lysate.  [00:05:00] Which is about what is left.  And so on.  But it’s worth commenting that there was an undergraduate who spent all her time making the various freeze-thaw lysates and sonic extracts.  Earned her way through college partially making these extracts for us.  And I’ll have to find her name.  It’s LB.  So here is LB dash 12 freeze-thaw lysate.  LB is Lisa Becker and she was an undergraduate.  I think she was a daughter of a professor if I remember rightly who spent all of that summer — [00:06:00] of that period of time, it wasn’t summer at this point — making these freeze-thaw lysates and sonic extracts for the packaging.  There she was a fun person.  And she’s thanked in the final paper for making these.  So LB12 is Lisa Becker’s package — preparation 12 of freeze-thaw lysate.  So at least that number 12 shows how many she’d already made.

And page two says in this particular example that it was adequate but not very great result.  Ligating the material ready for packaging.  [00:07:00] So just getting ready to do the experiment again.  More Xba fraction being made from P31.5B DNA.  Might be phag beta.  I’m not quite sure.  Back to ApaI again for the testing of integration.  Here is a map at least showing where the ApaI site is.  That there’s an ApaI site between delta and the DNA — for mammalian DNA, human DNA, between delta and downstream of delta and upstream of our [00:08:00] hopefully integrated fragment of DNA.  With a comment that unfortunately ApaI cuts pSV2neo once as well.  So the experiment becomes complicated.  It’s no better than Xba.  So that is a note that perhaps helps a little bit understand why we were interested in ApaI.  So preparation of ApaI- Charon 30A and the possibility of getting a Charon phage which didn’t have an ApaI site was being considered.

[00:09:00] More purified DNA to confirm that the targeting had occurred in the mammalian cells.  So here we are preparing material from k5 Xba and looking for the slow, medium, and fast fractions with a conclusion that SFF is just large enough.  [00:10:00] M may be too small.  But better pool M and S, etc.  For the 11-kilobase test.  Still wanting to be sure that things work.  So here we go back now to packaging again.  One of these complicated experiments like the page 139 experiment.  Packaging of k3 of kappa — that’s really kappa 3.  Of kappa 3 ligation.  Total C-1a was 145.  And the total number of phage was 4.2 times 10 to the 7.  So that it was a rather satisfactory result.  [00:11:00] With the secret ingredient again at the bottom of the page that they had — that in order to get the bacteria that would make decent plaques 3.6 milliliters of C-1a fresh overnight was allowed to — was diluted and given 15 minutes at 37 degrees in order to get it exponentially growing.  Bacteria on which to plate the bacteriophage.  So that’s the bottom of that page is really is a continuation of the message that was [00:12:00] anyway hidden on page 139 of book iota.

And in this case let’s see what the result was.  I don’t remember what the result was.  But there is the comment that the plaques are easily countable at midnight.  No note of positive, however.  That was [00:13:00] continuing production.  Testing for positives.  Looking at ligating kappa 7 fast fragment on Sunday, February 17th, page 11.  And here we go to packaging of that on page 13, Monday, February 19th, with a comment of bust.  One of the two number of phages was only 2 times 10 to the 5th.  Compared with the previous page which is 4.2 times 10 to the 7th.  So no wonder it was a bust.  [00:14:00] Only one plaque on C-1a with 2 times 10 to the 5th phages.

So continuing the same struggle Monday, February 18th, page 15.  Basically not a useful experiment with a comment repeat the experiment with a better freeze-thaw lysate.  So the freeze-thaw lysate maybe was a problem.  It was not always easy because this one on page 15, Monday, February 18th was on film but negative.

So looking at different freeze-thaw lysates and different buffers on Wednesday, February 20th [00:15:00] to see which was a better lysate.  But the conclusion is you could use LB42 with buffer A and buffer N1, etc.  This what are the best preparations and buffers to use.  So M1 buffer being made again on page 19.  And the prep gel.  Once again a prep gel of small, medium, and fast fractionation of kappa 5 Xba digest.  And continuing with kappa 21F ligation page 23 February 21st.  [00:16:00] And package.  And the result of the usual type of experiment.  These difficult freeze-thaw lysate packaging experiments on Friday, February 22nd, page 25 with different DNAs.

Again with growing the C-1a overnight and then freshening it up by giving it 15 minutes at 37 degrees prior to use.  And here the average Charlie B.  So here is — [00:17:00] we came to — on the left-hand side.  Average by Charlie.  Charlie, that was Charlie Blystad who did the titration.  He was a technician.  Was 1 times 10 to the 8 bacteriophages per microgram which was quite reasonable.  So these titers are per milliliter.  So to get to the total you had to multiply.

Hybridize to the beta IVS probe and to the 5.2-k probe as appropriate.  [00:18:00] Ligating the material.  We’re using the kappa 27 on kappa 27 the ligation of the different fractions.  This was without prefractionation.  So Saturday, February 23rd, page 27.  This was to [00:19:00] take the total DNA unfractionated, then test for direct ligation again without prefractionation.  And between 13 and 25 — 30 5.2-k-positive but no positives for the beta IVS so the conclusion was that not much insert.  The ligation was good.  Must have centrifuged out quite a lot.  Nick translating the 5.2-k probe.  Page 29.  Here we are back to the experiments again of the big preparations.  Always freeze-thaw lysates and [00:20:00] repackaging and so on.  Sunday, February 24th, page 31.  In this case 1.7 times 10 to the 8 total phages, 13 clear plaque-positive cells on 5.2-k.  That’s 18%.  But none were positive with beta IVS2.  So we’re getting — we were rescuing the supF but we were not finding the recombinants.

So comment.  The total C-1a properly screened to date is about 300.  The total 5.2-k-positive are about 40.  [00:21:00] And with two, three positives.  So more preparative gels of the size fractionation of the DNA on the following pages.  And with February 26th Charlie will repeat the ligation that was done on kappa 27.  The ligation is excellent.

So here we go again on page — so many times repeated.  Page 37 Wednesday, February 20th.  Another big packaging experiment.  In this case total number of phages was 1.1 times 10 to the 8, 100 had picked up the supF gene and so would grow on C-1a.  [00:22:00] But in this experiment nothing were positive with either the 5.2-k probe or the IVS probe.  So the conclusion was better make some new probes.  Pooling Cosos again on page 39.

Here we are back to sib selection with Ron Gregg’s material.  Start on subdivision of the 4,000 colonies.  So there were four preparations which [00:23:00] contain as I recollect something on the order of 400 colonies each.  And they were called 1B, 2B, 3B, 4B kappa 41 subdivision.  These were ligated each one separately into delta — into the phage DNA and Cosos pool ligation was also done on the same page.

So Saturday, [00:24:00] March 2nd, page 43, reviewing the situation of iota 139F versus kappa 33F.  The biggest obvious difference is that the C-1a on iota 139F was freshly boosted.  This is now realizing what was perhaps I’ve already assumed I knew but it — this is the very important point that the biggest obvious difference is that the C-1a on iota 139F was freshly boosted.  Therefore test this as a general procedure.  Test 3-1 C-1a overnight diluted [00:25:00] and allow to grow to an optical density of 1 in about three hours.  Being a very deliberate and clear attempt to get exponentially growing cells with a known optical density.  So this is really key to getting success.  With some calculations about how many bacteria there would be.  If there were 3.6 ml of this exponentially grown [00:26:00] C-1a there would be about 10 to the 10 bacteria, which is more than adequate for 10 to the 8 bacteriophages.  So this was a multiplicity of infection of 0.1 which was a very safe multiplicity of — so it was clear that this was the page where I really fully understood what was going on because on page 42 it says this looks like the way to go.

Packaging and plating on page 45, Sunday, March 3rd is now regrown C-1a from Saturday was used to set up the experiment.  It worked.  [00:27:00] Clear result.  The 3B has two IVS2-positive phages.  And 2B may have one nearby.  And 4B and 1B are negative.  Establishing clearly that the positives were in the right fraction of DNA.  So page 44 there opposite plated with no particular problems, etc.  Beta IVS2 and so on.

[00:28:00] Total C-1as on all of them were quite good, 1B, 2B, 3B, 4B.  All of them had several hundred.  Or in fact over 500, 500 to 900 C-1a-positives.  And here is a clear result that 3B has two IVS2-positives, and 2B may have one as I was saying.  Picked and retested, both confirmed as positives.  So this procedure allowed very clear results.  Cosos kappa 39F packaging on Monday, March 4th, page [00:29:00] 47.  But negative on IVS2.  About 90 on 5.2-k probes.  So no targeting.

Fresh probes being made Monday, March 4th, page 49.  And the repeat of the experiment 51.  Need to confirm that these are truly negative.  Those two preparations which had the fragments — the fractionated DNA where the mammalian DNA was greater than 10 kilobases long.  And so would have [00:30:00] the unmodified beta-globin region in it but would not have had the recombinant fragment.  Need to confirm that they were truly negative is the experiment on March 21st.  Preparative gels again on the following page for 3B, kappa 41 3B.  And just to remind what 3B is, 3B was just one of the preparations.

[00:31:00] Eluted as usual page 52.  And kept the gel samples.  Fast, medium, and slow.  With size ranges well shown.  Packaging the DNA then on page 55.  Friday, March 24th.  Got plenty of plaques.  [00:32:00] Yeah.  The results of this experiment are not given until page 60, Sunday, March 24th, thereabouts.  [00:33:00] Say the results of kappa 55 are that there was one positive on kappa 53, M, seven positive on F, one positive on 1B kappa 51, and no positive on 4B kappa 51.  So the results are being presented there.  Hybridization and nick translations of the probe are talked about on page 60.  Cosos repeat unfractionated on page 63, Thursday, March 28th.  Sloppy gel but the ligation is usable.  [00:34:00] So here we are packaging Cosos kappa 63 on page 65 with a conclusion that the 1,604 IVS 5.2-kb IVS probe are positive on C-1a but none positive with the beta probe.  None positive with the IVS2 beta probe.  So on page 64 it says that more than 300 of Cosos have been screened and none are positive.

[00:35:00] Nick translation on page 67 with comment that some summary of material on April 4th on various — give some account by me.  And probable and possible.  With Cosos.  [00:36:00] So on Monday, April 1st, page 69 review of the phage DNA stocks for the next round.  And on the following Friday, April 5th, page 71 start of the next round.  In this case this is material from Raju.  Because we’re looking at Cosos 117.  I think it’s 117.  But anyway from Raju RK79 through 84 DNA [00:37:00] was looked at to see how the digest with a comment that there are nucleosomes present because of repeated fraction sizes.  Also on the opposite page some film counts of previous results from Charlie Blystad.

So ligations of Raju’s DNA with a conclusion that a lot of [00:38:00] unligated query degraded DNA.  Must have been many dead cells in the original prep but continue.  So packaging now kappa 73 ligations which are these ligations with Raju’s material into Charon 3A delta delta X.  So that packaging now on page 75.  With the conclusion that there was a [00:39:00] reasonable number of 5.2-k-positive plaques but all are IVS-negative, so no positive result in terms of target.

Continue with Ron’s experiments or Ron’s DNA.  He’d extracted material but didn’t digest again.  So here’s back to work again on packaging experiment.  This was [00:40:00] Friday, April 12th, page 79, highest ever percentage of IVS2-positives.  C-1a, there was something on the order of 0.7 times 10 to the 6 phages, of which 149 were positive for 5.2-k and 3 or 4 were positive for the IVS, meaning that they were correctly targeted with preparation kappa 77 RGB11.6 were positive.  And negative were [00:41:00] the plaques obtained from RK83 repeat.  So here is the conclusion at the bottom of the opposite page.  Three are sure, one is probable for IVS2 RGB11.6.  So we’re now getting very much clearer positives.  At last getting to individual cells.  Since we know that RGB11.6 has positives and it is not a large collection of cells he is going to — Ron will clone individual cells of RGB11.6 and give them to me [00:42:00] in DNA pools of 20 or 50 or 100.  In other words carrying out the sib selection.  Expect positive in less than 5 pools of 20.  But in fact as we see below it was in the first pool of 20 that had contained the positive.  As we will see a number of pages later, kappa 117.

So here we are, kappa 81 at the present time.  Packaging of kappa 81 RGB31.5 of which a total of [00:43:00] 0.4 times 10 to the -6 of the total C-1a three positive.  No, this was some mistake there.  As commented on.  A loss, a mistake, on the ligation.  Mistake on the ligation was too great for that experiment on page 83.

Some more DNA from Raju Wednesday, April 17th.  Ligating it to the [00:44:00] Xba-digested phage DNA.  But some mistakes.  Again commenting on Raju Kucherlapati 85 and 86 ligations.  On page 87.  April 17th.  The conclusion adequate ligations but not very good.  So packaging again on Thursday, April 18th.  [00:45:00] With RK85, etc., etc.  And so hybridizing to 5.2-k and hybridizing to the beta IVS let it as usual, etc.  But not happy with the experiment.  Too low a yield of phages.  So this DNA is too poor is the comment.

So bacteriophage for the next round.  April 23rd, page 91.  On with it.  Doing similar experiments of Charon 3A delta delta X digested with Xba.  New DNA samples from Raju on page 93, [00:46:00] only a modest improvement.  If not good proceed to fractionate.  So we’re continuing with the packaging, page 97, Thursday, April 25th, packaging Raju Kucherlapati’s various preparations, 79, 83, 85, and 86.

[00:47:00] Onto film.  On Friday.  And off on Sunday.  But no comment on the results at this point.  Repeat on page 99, Friday, April 26th, repeat of Ron Gregg’s B313B material, [00:48:00] very suspicious pattern and bad cutting with the nucleases.  Continue but don’t expect very much.  So the usual prep gel to get the slow, middle, and fast fraction.  Separations were poorer than usual.  Page 100, Monday, April 29th.  F has a lot of S in it for some odd reason.  But M is usable.  Use all of it.  So Ron’s preparation.  Yeah.  On Tuesday, April 30th, page 103.  [00:49:00] Packaging the first on page 105, Wednesday, the — packaging Ron’s material.  Very odd.  Nothing — was difficult to see.  Only one visible at 8:00 p.m. of the plaques.  So not clear why this was giving poor plaques.  Because it had been grown up.  The C-1a had been grown up in the ordinary way.

[00:50:00] More packaging of Raju’s material on page 109.  And more fractionation of Ron’s material DNA on page 111, Friday, May 3rd.  Conclusion good cut.  Proceed if necessary.  Meaning that I thought I had the positive.  But am ready to do more if necessary.

So here we are on page 113, Saturday, May 4th.  Next round on Ron Gregg’s [00:51:00] RGB11.6 from kappa 77.  About 50 cells per well.  And so there were three pools.  Pool one 144B, 144A.  So 144 pool one, 144B, 144A.  And he thought that RGB144 pool one had about 20 single colonies pooled.  And so the question is whether the pool of 20 had a positive in it or not.  Or whether the other pools had positives.  And the result was Xba digestion of the materials.  And [00:52:00] ready for ligating them on the following page.  Pool one there being one of them and all ligated satisfactorily with the conclusion proceed.  Page 115, May 6th.  May 7th, then packaging these materials.

With a result on the following page that 43 phages were 5.2-positive, 5.2 IVS-positive in fraction 43, in fraction 62.  No.  Sorry, that’s wrong.  That’s [00:53:00] testing 144A, 144B pool one and 3B slash M the whole, etc.  There were four different DNAs being tested from four different pools.  And pool one had about — had 115 5.2 IVS-positives — 5.2-positives and 8 IVS2-positives.  So the ratio of 5.2 to IVS for the pool one was eight.  So eight clear positives obtained in pool one.  Just backtracking a little.  I might have occasionally referred to it incorrectly.  The 5.2 overview IVS means [00:54:00] that the 5.2 says that you’re positive for the supF and etc. and that the IVS means that you’re positive for beta.  So IVS is the number of correctly targeted plaques.  So here is a very important result then that clearly eight positives.  On the left-hand side, page 116, there’s a very big squiggly comment.  This is it, exclamation mark, greater than or equal to 8 positives [00:55:00] from about 30 IVS2 — 5.2-positives.  So something on the order of 30 phages had picked up supF and of these 8 were also positive for the IVS2 probe which means that they had been — contained the recombinant fragment.  So again at least eight positives on this page, probably the most important or the most positive result obtained with the packaging work.  So those two pages are rather a treasure.

And the following page [00:56:00] rather nice glider comment, I had the silver distance, not very far in my Schweizer SGS 1 to Lake Koshkonong on the weekend of — presumably the weekend before.  Still religating with Lisa Becker’s freeze-thaw lysate, 51 now.  I’m remembering that back there we had 12.  So this is Lisa Becker’s fifty-first preparation of freeze-thaw lysate still religating some for further tests.  [00:57:00] Going on with various other DNAs on page 121, Thursday, May 9th.  But abandoned because pool one was positive so I didn’t need to continue.

Again page 123 Thursday, May 9th, on the opposite page.  Abandoned because pool one is positive.  So here we are summarizing a general view on page 125, Friday, May 10th.  The positives on pool one makes this series of experiments obsolete.  Just finish that and the other.  But happy enough now that we have a positive pool in order to be able to get the individual colonies.  So a little [00:58:00] tidying up going on.  Page 129, Tuesday, May 17th, just trying some genomic tests on pools.  A bit early but I can’t resist the test.  These are Southern blots.  But nothing really convincing.  So still repackaging.  On page 131 [00:59:00] but this was with pool two ligation, pool three ligation.  And a reasonable number of positives for the 5.2-kb probe.  But none were positive for the IVS2.

Making some more nick translated probe for IVS2, Tuesday, May 14th.  And then we come to page 135.  Which is Saturday, May 19th.  And here are the results of the colony [01:00:00] of Southern blots using the material now from individual colonies which comprise the positive pool identified earlier.  So these colonies were — DNA from these colonies was prepared by Ron Gregg and he gave me the DNA to set up the Southern hybridization.  So these DNAs were from various colonies, 4, 6, 7, 8, 9, 10, 12, 14, 15, 18, and 20.  Different individual cell colonies.  [01:01:00] One gel was set up with low DNA and one with high DNA.  And run overnight by Ron.  And he was out of town.  And he gave me the privilege of blotting them and looking for the positive.  So these are colonies made with delta beta 117.  The simpler targeting.  And as is clear from the left-hand page 134, the number 20 is it.  The colony, the Southern blot shows fragment on the order of 8 kilobases length in that particular colony whereas all the others are 11 kilobases, 11 kilobases being the nontargeted size of the beta-globin locus, [01:02:00] and the 8-kilobase size meaning that the locus had been targeted.  So this was our positive by Southern blot.  Completely independent now of the phage assay.  And with the comment that it had taken three years and one month since the idea of doing this type of experiment.

And these are the results which were published in our paper very quickly thereafter.  The paper didn’t have any problem in being published and had some very positive results.

[01:03:00] So page 136 has a summary of the many experiments, the date and the page, freeze-thaw lysate that was used, the sonic extract.  What would have been — what the control was and what the C-1a-positive was to see what had happened.  So for example the top four experiments are with Lisa Becker’s freeze-thaw lysate 12 and 16 and then we go to her preparations B42 and Lisa Becker 49, Lisa Becker 69, Lisa Becker 51.  [01:04:00] And so on.  So at least there.  And the various sonic extracts.  But there were only two sonic extracts needed.

One was Mike Koralewski’s preparation F102 and Charlie Blystad’s preparation A57.  And just summarizing how these various preparations would work.  With a summary of fragment rescue from cells not expressing, EJ cells, or expressing the beta-globin locus.  [01:05:00] Which are the X11 or X311 chromosome cells.  The ones in tissue — in suspension culture.  So expressing or not expressing beta-globin summary of what cells were used.  EJ carcinoma cells without G418 selection.  And X11 hybrid cells with G418 selections, to experiments.  And so summarizing the data.  Getting ready to publish.  [01:06:00] OS final double-check.  The following page has then flask one, flask two, three, four, five, six from Ron’s experiments with the X11 hybrid cell and pool one, two, and three.  Showing how many phages were obtained.  How many grew on C-1a and how many were positive for the beta probe and how many were positive for the IVS2 probe.  So there it’s worth looking to reconcile this with what we talked about earlier.  Pool one, which is the third from the bottom.  That total [01:07:00] genomic DNA that was assayed was about 40 microgram.  Phages which grew on K802, that’s the total number of phages was 7.5 times 10 to the 7.  Of these 115 grew on — sorry.  It’s 115 grew on C-1a, meaning they had supF.  And of these 115, 36 were positive for the beta probe.  Meaning that they had picked up the beta-globin region and the supF — beta — no.  They contained beta-globin DNA.  And eight of these [01:08:00] were positive for the IVS2 probe and had therefore been targeted.  So again eight were targeted out of these cells.

So that’s a summary of the material which is eventually incorporated into the paper that we wrote.  Monday, July 8th, some ideas.  Page 139 and Tuesday, July 9th.  Thinking about tests of gene function delta left and delta right that we had already been — that I had already been working with Raju in doing this type of experiment.  [01:09:00] To see whether cells were inactivated in different ways.  And on Tuesday, July 9th on the opposite page thinking about gene conversion and expression.  Gene conversion being illustrated and gene conversion plus expression.  How would one do that type of experiment?

And Art Banks had plasmids that we were thinking about on page 141, Tuesday, July 9th.  [01:10:00] So this was pSV2neo mutant epsilon and pSV2neo with beta S.  Beta S being sickle.  Received from Art Skoultchi.  Art Banks, sorry, Art Banks.  About nine months ago.  And transformed with Nobuyo Maeda competent cells.  To rescue these plasmids.  pSV2neo mutant epsilon is fine.  But zero on the other.  And he suggested — we talked to Art Banks and he said try it on the kanamycin plate.  [01:11:00] No obvious result quoted.

But on Wednesday, July 10th getting toward the end of this book.  Page 145.  Kpn digest of the delta beta 17 vector.  With conclusion that the cell — this tube is mislabeled.  Start again.  So going back to something better.  Cla17 single-cut BstXI with Cla17 eta 119 [01:12:00] DNA.  Quite a long time back now to single-cut this Cla17, not Cla delta beta 17, Cla17, the large piece of DNA.  Conclusion considerable degradation but proceed.  So Kpn digestion, religation being taken up on page 149, with some technical comments.  Better do phenol hydroxyquinoline extraction first.  So here we are now transforming Kpn delta beta 17 on page 151.  [01:13:00] Colonies on all samples.  NZY ampicillin and NZYDT ampicillin.  So backup restart of Kpn delta beta 17 Friday, July 12th, page 153.  Again some comment.  Technical.  Next time use T4 polymerase instead of S1.

So minis made from kappa 155 ligation, which was [01:14:00] Kpn-digested material.  So the minis were made from that material.  First check all with Kpn alone, etc.  And with the conclusion that a nice set of minis.  But either Kpn didn’t work or there are no positives.  So extracted them on the following page, July 16th, page 159.  Extracted the first 16 of those.  There were altogether — I should have said there were 48 minis were picked on page 157.  And the first 16 were extracted with phenol hydroxyquinoline [01:15:00] on page 159.  And the conclusion.  Not as much.  That there was not as much self-digestion, but there are no Kpn cuts.  So what to do, use more BstXI.  Time and T4 polymerase to get the blunt ends.  And that ends book kappa.  [01:15:34]


[00:00:00] One of the very satisfactory books, because in that is the results of the experiment which basically was most important as far as I was concerned in getting the Nobel Prize.  Of course it was not the only thing that was contributing.  [00:00:19]


[00:00:00] Yeah.  This is — there were a couple blank pages between — in kappa 142 and kappa 143.  In there I was actually — was included some maps of the delta beta supF fragment.  July 9th, 1985, a map of the delta beta supF fragment.  And what was needed to do to use that fragment properly.  So for example on page two of the addendum it says plasmid delta beta 17 Kpn vector single-cut with BstXI, etc.  Blunt with S1.  And so on.  Different recipes that were needed.  [00:01:00] And attached to the thing are a bunch of papers handwritten, are some circular maps or computer maps of the P supF sequence checks from the computer.  So that’s where this insert belongs on page 142.  [00:01:37]