The new new cuisine

I came on this fragment of a transcript when I was working this morning. AC is Alan Coulson, at the Sanger Centre, who probably knows more about the extraction of DNA than any man alive. “John” is Sir John Sulston. ACB is me, trying to make sense of it all. Any resemblance to the girl who earns her living is accidental, and right at the end.


ACB: “What were the actual physical materials you were dealing with? Somebody takes a large quantity of worms, presumably a thousand or whatever?”

AC: “About a ml, probably.”

ACB: “A million?”

AC: “A ml. Volume. 1 millilitre. I’m not sure how many worms that is, actually. You’d have to ask John, I’m sure he’d know.”

ACB: “OK, a millilitre of worms and, basically, the first thing you do is grind them up?”

AC: “Yes, in liquid nitrogen.”

ACB: “Right. And then how do you go about – you’ve then got this goo?”

AC: “Powder.”

ACB: “Very, very cold. Freeze-dried powder.”

AC: “Well, you add, essentially, detergent and salt.”

ACB: “And that cracks open the cell walls?”

AC: “Yeah.”

ACB: “Of all the constituents of the cell, how do you then get the DNA out? Or does it not matter?”

AC:“You can extract it with phenol, which removes most of the proteins and other gunk and leaves your DNA in this rather pure solution and then you can precipitate the DNA out of that with ethanol.”

ACB: “Right, vodka, yeah…And there you’ve ended up with DNA as little damaged as possible and, presumably, the freeze-drying is part of the non-damaging bit?”

AC: “Yes. Well, it’s not freeze-drying but by keeping it cold, you prevent enzymic degradation, although, actually, we used a strain that is deficient in nuclease to minimise that.”

ACB: “And you then have a very, very small quantity of pure DNA?”

AC: “Well, you can get quite a lot out of the worm, a lot of DNA, that is.”

ACB: “How much?”

AC: “Not sure, have to look that up.”

ACB: “A visible – ?”

AC: “Oh yes, a big goop.”

ACB: “A big goop. Sounds like a very old-fashioned cookbook. ‘Take a big goop – a generous handful.’ So the big goop is then sorted into lots and lots of smaller portions?”

AC: “Well, then you digest it with restriction enzyme, very gently, just minimally, and run it on a gel and, of course, you have to assay this and make sure you’ve got the right level of digestion. Then run it on a gel and you get a band, a smear, on your gel. Then you cut out the size fraction that you want, about 40 kb, and then you could put that into your cosmid vector directly but, in practice, we did a second purification because there’s always some smaller stuff in there.”

ACB: “Hang on, I know I’m going very slowly but when you’ve run it, you put a little bit of restriction enzyme in? You’ve still got one tube full of goop?”

AC: “Well, you take part of the goop, you wouldn’t – ”

ACB: “Yeah, that’s what I was groping towards. So you split your goop into smaller portions?”

AC: “You just take the amount that you want.”

ACB: “And then the smaller portion that gets a little extra goo, which is the restriction enzyme?”

AC:“Yes.”

ACB: “And you leave it for a bit – a carefully measured time – in order to get restricted?”

AC: “Mm-hm.”

ACB: “By which time, it should all have been chopped up?”

AC: “It’ll be randomly chopped – well, it won’t be random but – ”

ACB: “The lengths will be fairly random. You take some of these out and put them on a gel.”

AC: “We take all of them, actually.”

ACB: “You take all the goop, put it on a gel, run it up, and then you said you cut it out, you actually physically cut out the gel and you’ve still got the DNA in there which has migrated a certain distance, according to how long it is.”

AC: “Yeah.”

ACB: “Right. And then you get it into bacteria somehow.”

AC: “Then you stick it into your vector – stick your worm DNA in there to re-circularise and then you – ”

ACB: “So you have a dishful of straightened-out circular plasmids which are looking to join up again and you add to this the worm DNA and where they join up, they’ve incorporated the worm DNA.”

AC: “Yes.”

ACB: “And all this is actually taking place physically by moving this liquid into that dish and this – ”

AC: “In tubes, yeah.”

ACB: “And, finally, you get those back into the bacteria and the bacteria then multiply like mad.”

AC: “And you take them out and each one of those bacteria which has one piece of the worm DNA gives rise to a colony. Then you pick those colonies off and grow them up and then freeze those in glycerol, which means that freezing doesn’t kill them. In fact, those clones that we made nearly twenty years ago, we’re still distributing to the community.”

ACB: “And what do they physically look like? You have a little petri dish with a sort of smear in the middle of it?”

AC: “Colonies? [Gets out something that looks like a white plastic postcard with a grid of black spots on it.] That happens to be a square one.”

ACB: “And each splodge is a colony?”

AC: “Yeah. In fact, that’s human: that’s John.”

ACB: “Bit of a conversation-stopper, yes.”

This entry was posted in Worms. Bookmark the permalink.