Scientific methodology seems to come in distinct phases for me. One month I’m knee-deep in biochemistry; the next it’s all confocal microscopy on cells, or annotating images onscreen. This is part of what I love about research: the familiarity of the manipulations coupled with the variety that keeps everything fresh.
Cloning panache Sometimes it’s cool to be old-skool
This week, it’s become clear that I’m moving into a distinct cloning phase. Of DNA, as opposed to of animals (or of an army of evil minions – although they might prove useful for analyzing my 20,000 movies.)
Now, this is pretty familiar territory – I spent almost my entire PhD stint cloning constructs. Things were pretty primitive back in the early Nineties. There was only one sort of Taq polymerase, and you purified DNA using phenol, or on cesium chloride cushions if you were really fussy. You made your own competent cells. You extracted fragments from gels using low-melting agarose. You sequenced your final product by hand – with radioactive 35-S. If you had something really huge to clone, like a retroviral genome, you had to clone it using lambda phage – oy vey, was that a pain. If it was small, you used M13 phage. (For my younger readers, imagine a tape cassette – this sort of palm-sized plastic thingie that could store about an hour of music. Cassette tapes are to MP3 players as M13 or lamda cloning is to the current methodology.)
In the meantime, some things have changed. And not all progress is unwelcome. Yesterday I made a construct using the Gateway system – I don’t like to give excessive airtime to product placement, but this nifty system does away with the classic cut-and-paste ligation in favor of a snazzy recombination job on vanishingly small amounts of starting material. (This means you can usually clone directly from your collaborator’s miserly gift of 0.0001 microliters of DNA without having to amplify first: bonus.)
“How many colonies should I pick for the minipreps?” I asked one of the youngsters in the lab. “Six? Twelve? Twenty-four?”
“How many?” He blinked at me, uncomprehending. “Just one. They’ve all got the insert, all in the right orientation. Otherwise they wouldn’t grow.”
Nice. I can live with that.
But other things, reassuringly, haven’t changed. In fact, my knowledge seems a sort of forgotten, retro-chic dark art that is coming in extremely handy amongst the younger lab denizens when their turbo-charged, kit-driven cloning doesn’t work the first time. Some of the tips I’ve passed on (to frank adoration) include using the Klenow fragment to help finish off the ends of PCR product, for when that swanky new brand of Taq gets too lazy to complete the restriction tail you’ve engineered in; ethidium bromide spot plates, handy for quantitating tiny amounts of insert and vector when the molar ratios really matter, as for three-way ligations; the concept of low-energy hand-held UV illumination that doesn’t damage your DNA fragments while you’re cutting them out. These are all things that I would have thought I’d long forgotten, but which seem to have become ingrained when I wasn’t looking. It is almost as if, after returning to the lab after my career break, I had to go through that distressing one-year period of almost complete lab amnesia before being able to retrieve all the stuff in long-term storage.
Kids these days? They just don’t understand.
Ah, yes, nostalgia. It’s sometimes handy to be able to show those young whipper snappers a thing or two. For example, when the World was Young, Dr M. C. of Kingston taught me how to mark up text for the printers, a skill which I (mainly) retain and which still comes in amazingly useful.
And I have another more tech-oriented story. One day, my then computer got infected by a virus whose source could be traced. The source told me they’d called in specialist help. I rang the aid specialists to ask if they’d come over and debug my machine, too.
“No,” they said, “you couldn’t afford us. We cost ninety quid an hour”.
I pestered them and told them that I’d pay what it cost, as their expertise stood between my machine and oblivion. They still refused to work for me. After yet more pestering from me, the man on the phone went all sotto voce and said, look here sonny, go to this website and follow the instructions.
So I did. The instructions were frighteningly techie but after I’d read them through a few times and taken a deep breath, I realized what had to be done.
The virus was 32-bit, so I had to go into DOS, which is 16-bit, and type in a few instructions.
As soon as I went into DOS I felt like I was coming home, and the rest was easy. Computer cleaned up in seconds.
Kids these days? They just don’t understand.
But for a saving of ninety quid an hour, it sure is good for us oldies.
bq. the concept of low-energy hand-held UV illumination that doesn’t damage your DNA fragments while you’re cutting them out.
I learned that one in … gosh, 1992 it would have been. Amazingly, my cloning started working.
The number of people I’ve known who used the high-power transilluminators and said ‘Oh I’ve never had a problem’, and then _they did.
I didn’t get taught any of this stuff in classrooms – I was taught by my PhD supervisor and postdocs. I suppose as more and more people use kits and outsource to do (supposedly) routine things like cloning, the fewer people there will be floating around your department who know the tricks. This is kind of sad, but when I use a kit I can understand the lure.
Mmm–sorry I gave the wrong impression. That trick was something I learned from a fellow grad student in the lab. No classrooms in UK PhDs.
Well, in America you get taught a lot of practical molecular biology as an undergrad – lab-based courses. But you only got to practice the basics.
Hah. UK courses undergrads are lucky if they come out knowing how to hold a Gilson.
Arthur C. Clarke said “any sufficiently advanced technology is indistinguishable from magic”. Perhaps we could add “in any hi-tech domain, any sufficiently primitive technology can be indistinguishable from magic”.
It doesn’t matter what the technology is… computers, biochem, whatever.
Yes, magic, kits, and machines that go “ping” are fine, until something goes wrong. If you’ve previously taken a couple of days slaving over a single-beam dials-and-needles spectrophotometer to do what the “ping” machine does in 10 seconds, then at least you have an idea about what might have gone wrong with the “ping” machine, and a chance at figuring out the workaround.
We’d have killed for something like Taq Pol PCR in 82 (Mullis was 83). Restriction endonucleases were the magical “kits”, we’d spin the fragments in caesium chloride gradients to separate them, put them in a beaker full of target bugs with lots of holes in their cell walls (watching the Ca concentration so they didn’t die), use a stirring rod to “integrate” the fragments into the target genome (hopefully, but who knew where), and then have to culture for a few days to see what came out. Then you could hazard a guess about the site of the gene in the original plasmids. Apart from the dial-up-the-volume micropipettes and centrifuge tubes, it was bucket biochem (if you use 1 litre glass buckets).
It might sound horrible, but we probably made up the time by not having to worry about dealing with ethics committees
Mind you, the neatest trick was how we integrated under the unknown curves on paper coming out of things like chromatographs or GRASS analyzers: cut the paper along the squiggles and weigh the pieces. I wonder how noughties students would approach that problem. (Scissors and scales elevated to dark arts!)
“the concept of low-energy hand-held UV illumination that doesn’t damage your DNA fragments while you’re cutting them out.”
That one holds true for T7-tagged PCR amplicons that you hope to use for synthesizing riboprobes, too. We learned the hard way, but it comes across as an arcane dark art when I transmit the knowledge to the next generation of youngsters who want to do an in situ hybridization and can’t get their labelling to be efficient.
Mooooaahahahaha…
If you’ve previously taken a couple of days slaving over a single-beam dials-and-needles spectrophotometer to do what the “ping” machine does in 10 seconds, then at least you have an idea about what might have gone wrong
Are you dissing my NanoDrop? Cuvettes are definitely something I don’t miss about the good old days.
David, that’s fascinating — and I adore the idea of weighing paper to work out the area. I guess things seemed a lot more fun when we could poke and prod the methods, as you say. Although I have to point out that I’ve just used LyseBlue for the first time today — a dye that tells you if all your plasmid prep lysate has been properly neutralized, by turning from blue to white! It’s like something out of Harry Potter – and definitely something to trot out when the TV cameras come round the lab.
NanoDrops and “cold, dead hands, prising out of” go together in my book.
I don’t get LyseBlue though, at all. goes off muttering
It’s lovely. Sort of a cobalt color, and then it goes all squidgy with the SDS precipitates — reminds me of storm clouds. You have to take poetry where you can find it.
Remember, Richard had a painful stint at a company that subsequently slipped that very product into their prep boxes. But I agree, it’s pretty.
heheh.
No Heather, ‘my’ company didn’t have the blue dye. Pretty sure they stopped making the miniprep range when they were bought: I did make disparaging remarks about a certain over-priced company beginning with ‘Q’ and their claims of innovation, though.
There are about a hundred dusty old blue boxes of products from the company beginning with Q in our lab — nobody knows how old they are or who used them last but we are superstitiously afraid of throwing them away. They give you far more buffers that you will ever use up, so it seems wrong to chuck them — the Q gods might get angry and curse your midiprep.
Having said that, when the Alan Hall lab decamped, they left behind a fine collection of old restriction enzymes (including the really weird ones with names longer than 5 characters). We still use these, even though some of them are dated 1991.
I was afraid to use LyseBlue for the longest time (Why would I add an ingredient to something that already works?) but then my own bottle ran out and I had to use the lab stock, and – ooooooh, it was pretty!
shakes head
It’s pretty, Richard, PRETTY AND BLUE!
Actually, it kind of looks like my avatar. That’s actually a cartoon of Bromophenol Blue (Advertisement! ), but maybe that’s what LyseBlue is. I don’t know what chemicals I was throwing in my preps.
Hee hee. The point is, Eva, that such–
oooh! Look! Shiny!!
Dyes are good. Anything that gets us away from the reputation that all we do is pipette tiny amounts of colorless liquids from one tube to another.
Even if it’s true.
More explosions, I say. Henry?
Hey, I’m the one famed in song and story for explosions. Did I tell you about the time I nearly burned down a school chemistry lab?
“They’ve all got the insert, all in the right orientation. Otherwise they wouldn’t grow.”
Ohh, careful… As someone who is doing a lot of Gateway cloning (think 15k clone library construction), I can tell you that they often grow with the wrong insert in an apparently completely random orientation. That’s the one problem with recombination, it’s not 100% predictable (more like 99.9%). I always pick at least 2, usually 3 colonies to check. It is a huge step up from the old school ways of cloning though, my project would be untenable without it!
ooh…thanks for the tip. Unfortunately I’ve already done my midipreps – can already see on the gel that one looks slightly smaller than all the others. (Can they grow with no insert at all?) But I’m the sort that’s likely to go for at first, then go back and do it properly if the luck doesn’t go my way.
I’ll pop them into human cells and see if any of them glow! All part of the fun.
Picking even 2 or 3 clones per construct would have saved me sooooooooooo much time during my PhD and postdoc… when I was in full-on promoter mutation mode I’d be making 10 different constructs, and picking 12 clones per construct to have good odds of getting the right insert, in the right orientation, with the right mutation, on the first attempt.
24 minipreps (a rotorful) per day, every day, for a couple of weeks… not enough money for Q kits… good times.
And it was uphill both ways, in the rain.
@Jennifer they regularly (but not too frequently) grow with no insert, as long as the plasmid backbone is there and the ccdB gene has jumped out they’ll grow.
@Cath I learned to do them the “cheap” but slow way too. Now I miniprep multiple 96-well plates in day. You don’t want to see the pile of discarded plastic (~8 tip boxes per plate) at the end of one those days… or the sequencing analysis at the other end
Colony PCR, you know you want to.
Actually, I’ve been know, when using GFP, to not bother with restriction digests but just to go straight to transient transfections in a 24 well plate and look for glowing cells.
@Richard: As I said, that was the plan: I’ll pop them into human cells and see if any of them glow! All part of the fun.
There are apparently lots of miniprep robots out there but I’ve never worked in an institute where that was an option – at least not a free option.
They’re called summer students. We file them under ‘disposables’.
I”m not sure I’d trust a summer student with my minipreps. I once had one who couldn’t seem to grasp why one had to change tips when pipetting into different tubes.
Ooh, that reminds me of a summer student we once had. Blog post brewing…
There is a legendary and terrifying tale in my old lab about a summer student.
She was there before I came. There were two witnesses (who I do know) who both have a history of not conflating stories, so it must be true, but it sounds absolutely unbelievable:
One day, in the lab, the summer student at the time was in tears about something. Completely upset. The lab tech asked her what happened, and she had accidentally stabbed her arm with a needle that contained some salmon sperm DNA. “That’s not so bad,” the lab tech said, “do you want a band-aid?” To which she answered, “What if I’m pregnant?!”
From a needle. In her arm. With DNA. From a fish.
Yes, really.
Epilogue: she got into med school. And this was long enough ago that, yes indeed, she is probably out there practicing medicine as we speak. Terrifying!
[Sidenote: Richard, did I do the quotes/commas/caps right this time?]
Excellent story, Eva.
Regarding pipette tips, there is, somewhere, a newspaper photograph of veteran geneticist Kay Davies holding a Gilson, ready to plunge it into a tube of something or other, with no tip on it. So I was told by one of her previous DPhil students, anyway.
Jenny, you’re making me all misty-eyed for S-35 sequencing on slab gels. That was something I was really good at. [sniffle]
Boggle.
{puts on Lab Lit Fiction Editor’s hat}
um.. yeah.
Did you de-gas your acrylamide, Richard (W)? I always loved that bit.
Eva – that is so funny! The scariest summer student I ever saw was in the lab in Leiden. She was assigned to the most mild-mannered, easy-going post-doc, but all you could hear across the lab was the post-doc exclaiming, every five minutes, Wat doe je daar? …Loosely translated, I presume, as What the f**k are you doing, you ***ing idiot?
Great stories!
I once saw a video of Michael Smith) showing some journalists round his lab. He wanted to show them a DNA prep, so he took over an in-progress prep from one of his trainees, grabbed a pipette, and almost started mouth-pipetting! He only stopped when the whole lab shouted “NO!”
I once got a mouthful, in a university lab practical. Potassium chloride, if I recall correctly.
I’ve never even tried!
De-gas the acrylamide? Hm, I remember doing that early on, but by the time I was a post-doc I just didn’t have to… bubbles? Never. Easily removed by the time-honoured “whacking the glass plates with the handle of a Gilson” trick.
I once caught an undergrad student very carefully measuring out and drawing evenly spaced diagonal lines on masking tape with a black marker…. classic
howls of derisive laughter, Bruce.
@Darren I believe our mutual friend Dr PT once did that in Glasgow. He thought it was done to denote the boxes of tips that you had to use more carefully.
I’ve never seen him do anything carefully, except maybe trying to avoid spilling his beer
I did say more carefully
Oh, bless. And you wondered why your autoclave seemed to suddenly have stopped working properly…
laugh
remind me to pull out the photos of when someone didn’t add water to our old autoclave…
The drawing of black lines on the masking tape was actually done in lieu of autoclaving, so no equipment risk was incurred…
At least she was smart enough to actually autoclave the things first, then stick the tape on later to denote the sterile ones.
If you want stupid I could tell you about the 2 undergrad students who spent 2 hours working in a hood… with the UV light on. Well, you don’t want any contamination do you? They had awesome suntans for a few weeks, and the nasty retinal inflammation went away after a few days with some powerful eye drops.
muppets
At least she was smart enough to actually autoclave the things first, then stick the tape on later to denote the sterile ones.
Ah… now, you see, you’ve just shattered my illusions.
Our most recent summer student was using this very expensive multi-well wand aspirator for washing out her 384-well plates — and then threw it in the bin when she was done. By the time anyone noticed, the trash was taken away. When queried why she would assume an obviously highly technical piece of equipment comprised of acrylic and metal parts was disposable, she replied: “Because everything else you use in tissue culture is.”
Oh dear.
Put her on glass pipette washing duty.
She broke them all.
You know, I’m not surprised.
In some ways this is rather unfair — I myself was once ‘one of those stupid summer students’. But my series of awful mistakes probably deserves a blog post in its own right.
You learned though. At least, I assume you did.
We had a trophy in our lab (made from garbage, aluminum foil, pipettes, etc.) especially for stupid mistakes, so people could see when the thing was on your desk that you had connected the black wire to the red thingie or something.
In my first postdoc, the lab had a Golden Pipette Award for the lab member who had the best data – the sort of person for whom everything always worked. It was an actual Gilson, spray-painted gold and mounted on to a piece of wood.
Needless to say I never won it.
that you had connected the black wire to the red thingie
I once blew the fuse in an electroporator by doing something like this. It was an $800 fuse. Or maybe a $0.20 fuse and a $799.80 labour and shipping charge.
Then I left that lab and didn’t touch cell culture for the ensuing seven years.
Hmm… do you have a German accent, ‘Richard’?
“Richard” – means “brave power”, derived from the Germanic elements ric “power, rule” and hard “brave, hardy”.
Oops, said too much.