Main

I'm going to a Gordon Conference at the end of the month. That's essential a pretty hardcore scientific conference, with a full programme and lots of very clever people in attendance (and me). This particular meeting is at Colby College, Maine, which is in the middle of bloody nowhere.

Seeing as I'd have to break the journey anyway (because trans-Pacific flights arrive on the Eastern seaboard at stupid times), and overnight either in Boston or New York going up and coming back, I've decided to take an extra day and do the tourist thing. It doesn't help that the conference finishes on the 4th July, which is some kind of public holiday or party in the US. I might see if I can see fireworks anywhere and ask what's going on in my best English accent.

So. It took me about half an hour to write the abstract and a similar amount of time to create the application online. The Black Queen spent Thursday afternoon looking for flights, then I took over and spent all day Friday trying to juggle connections and whatnot, finally making bookings Friday night. On the weekend I rested (actually, I didn't: but that's a story for somewhere else). Monday morning I figured out the hotels I'd need, and since then I've been banging my head against the completely arcane and impenetrable accounting system in order to get approval for the money I've already spent on the corporate card for the flights, and to make a claim for the hotel bills that have to be paid in advance and have had to come off my own credit card.

It's a bloody nightmare. You've got to do all these things in the right order, and you can't click 'save' until you've got all the information: if you hit 'submit' too soon it shoots off to get approved and you have to winkle it back out (easy to cock this one up — the buttons aren't labelled in any meaningful fashion) — and if, because you like to multi-task, you decide to open a claim for one sort of expense while working on a travel requisition then the second opens in the first window (not in a new tab, no, that would be sensible) and you lose everything you've done up to that point because you can't hit save yet.

My travel requisition might be a little terse for that reason.

Anyway, I got it done, and if I haven't checked all the right boxed or filled in every last detail I'm sure someone in admin will scream and shout. Fine.

After all that I went into the lab and buffered some phenol/chloroform. It was comforting. The semi-ester scent of isoamyl alcohol purified my senses. When people saw me in labcoat, gloves and safety specs they knew I was doing something dangerous. And real.

It felt good to be alive.

a-z.jpg

The arrangement of chemical stocks on shelves is important. But what causes arguments is whether it should be alphabetical by common name, or alphabetical by chemical symbol.

Obviously for some things — amino acids, or Tris and MES perhaps — the common names should win. However, and the first labmate who could immediately recall the mass of sodium chloride agrees with me, sodium chloride should be under 'N'.

We both automatically look for potassium salts under 'K'. Et cetera.

Wars have been fought over less.

A common technique in a structural lab is to grow up a protein in bacteria and purify it. It's pretty much essential to all structural work (NMR of packed bacterial cell pellets is a different matter, and We Don't Do That). To make things easier for ourselves, some people like to grow 'their' protein fused to something else that makes purification easier.

For example, a protein that we'll call GST binds to a chemical known as glutathione. If you can immobilize glutathione on some sort of supporting matrix, then you can use this to pull GST out of a complex mixture of cellular protein (pretty specifically, in a lot of cases). So if you make a 'fusion protein', consisting of your protein linked to GST, you get a quick and simple way of purifying the thing you're after.

A complication arises if you then, as we often do, need to separate the GST from your protein. We do this by using specific proteases, that you can think of as a pair of molecular scissors which 'cut' the fusion protein at a certain place. In this example we have engineered (yes: "genetic engineering". It's What We Do) the fusion to contain this 'certain place' between the GST and your protein. So in theory you can bind the fusion to the supporting matrix/glutathione, treat with the relevant protease, and obtain just your protein.

There are, as ever, technical issues and nothing's ever that straightforward, but this is what goes on all the time in my lab.

Now, one of the proteases we use has the name human rhinovirus 3C protease. A certain company sells it to us under a tradename, let's call it "Scissors"™. Buying "Scissors"™ is expensive - it works out at around Aus$60 per prep. So the Black Queen finagled a plasmid from someone, worked out a method, and now makes our own "Scissors"™, for about a tenth of the price. Thing is of course that Certain Company gets really upset if we say "Scissors"™ — in a publication or whatever — and didn't actually buy it from them. BQ has been telling everyone that we can't call it "Scissors"™.

"Human rhinovirus 3C protease" is rather a mouthful, though: It's a lot easier to ask "Where's the "Scissors"™" than "Where's the human rhinovirus 3C protease?". To keep everyone happy, we've come up with a new name for it. We're now referring to human rhinovirus 3C protease as K-Zyme, which is easy to remember, say and type, and isn't a tradename. So anyone can use it.

And if you would like some K-Zyme, and instructions on how to make it, then apply to the usual place.

I have been meaning to have a whinge about the scientific kit culture for more than a little while now.

Last year some time, I used a Qiagen miniprep kit (what can I say? I was in a hurry) and found that some noddy had added the blue dye to one of the reagents. Now, some of you will know what I'm talking about, and require no further explanation. The rest of you; seriously, you're better off not knowing.

The point is. . . well, no. Let's step back a bit.

Just over eight years ago I left a DNA extraction technologies company for reasons that do not concern us here. Eighteen months before that I had completed my first project at that company, which was to revamp a range of DNA extraction kits — minipreps, gel extraction, that sort of thing. I compared my efforts with those of the market leader, Qiagen (Promega and the rest, sorry, but even if your stuff is better it's not the benchmark). And even if I do say so myself, I did a pretty good job. My miniprep kit was faster, gave better yield, as good if not better quality, and was cheaper to produce.

It was, naturally (like the rest of the company down the line), torpedoed by a worse than useless sales and marketing department, and intellectually challenged management.

But I realized, last year as I used the Qiagen kit, that in more than eight years the market leader had failed to innovate at all. Minipreps are pretty critical to molecular biology research, and they still charge a 500% markup for something that, frankly, is crap.

Qiagen's idea of innovation is to add a blue dye, so that people can tell that something is mixed thoroughly. Which is bizarre to say the least; doing minipreps is one of the first things you will learn in a mol biol lab, and if you can't get that right first go you're going to have much more fundamental problems. Qiagen did nick one of my ideas, which was to add a collection tube to the kit. Now, I persuaded management that such a tube in the kit would be a good thing, and moreover, it should have a lid with an extended hinge so that it would actually fasten over the spin column while in the microfuge. Such a tube exists (and existed back then), so we just bought a job lot and packaged them with our stuff.

So Qiagen can't even get that right: Their collection tube is un-lidded, so you still have to fart around with different tubes at the end of the prep. To be fair, they did drop the five minutes on ice thing from the protocol, so I guess someone must be awake there.

Now, you might think I'm being hard on Qiagen, and you'd be right, but over the weekend I received the following (edited) email from my mate Tiddles:

Qiagen Maxi-Kit. first run = 1.3µg DNA/µl = ace. 4 times since ~0.3µg DNA/µl = a fornicating waste of my time. Fornicate. Fornicate. Fornicate. Fornicating fornicating kits. I fornicating hate fornicating kits. I have changed kit, changed solution, re-transformed my plasmid and still bollocks. Do you know what they think it is? I actually agree. . . It's growing too fornicating well and thus clogging their miracle fornicating filter system. What the fornication? Growing too well? What am I supposed to do? Put fornicating bleach in my media?
and this reminds me that not only is Qiagen's miniprep kit crap (but that's OK; when you're the market leader and everyone buys your stuff you don't have to improve or innovate, you just laugh all the way to the bank. Hmm. Reminds me of another company; one that starts with 'Micro' and ends with 'soft'), but their maxiprep kit (which, if you don't know and can't guess, is used for making much more DNA than a miniprep can) is even more execrable.

It's expensive, time-consuming, uses an imperial shedload of plastics, doesn't actually yield all that much DNA and, as m'friend Tiddles points out, can not cope with rich media. So to prep a lot of DNA for, say, multiple transfections, you need to do multiple preps. And if you try to use a decent amount of culture (like 101 ml instead of 100 ml) or rich media (2xTY) instead of that anæmic LB muck, the (bloody expensive) column throws up its beads in despair and fails miserably. Just like the Qiagen miniprep, in fact.

Y'see, back in '98, my miniprep kit could cope with buckets of rich media and still spew out loads of top-quality DNA, so I am very down on Qiagen for not coming up with something better.

But so that you know that I can do so much more than just whinge, my very next post will contain a link to the protocol that I currently use for maxiprep DNA. It makes loads, uses phenol, and gives you the feeling that you're doing some real chemistry. Great stuff.

Drive

7 December, 2007

So what do you do when you're learning to drive a (different) confocal microscope, it's half past 5 on a Friday, and you're only halfway through a 78 image Z-stack?

That's right, you muck around on the internets.

Failing that (because I don't know the password to the USyd cache and so can't actually get out of the building. . .) here's a random picture of what I'm doing:
Blue and green stuff

No, I don't know what it means either. Damn, but I need a beer.

This is a message for local people. . .

Dodging the rain on the way to Redfern last night I found an eppie:
eppie.jpg

It says

EcoRI-IL6 150 ng/µl 11/'07

on the side and
lin-IL6 150λ

on the lid.

At a guess it's linearized IL-6 DNA. If you want it back (long shot, I know), give me a shout. I want to know where you get your labels from if nothing else.

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My Beloved Monster

16 November, 2007

Coincident with the safety audit, there's been a bit of a discussion over at the Science Advisory Board about ethidium bromide, 'safer' alternatives and other ploys used by unscrupulous marketeers to get you to buy their company's product.

Ethidium bromide (EtBr) is a chemical that is used to 'stain' nucleic acid (DNA, RNA) so that we can actually see it in gels. Because EtBr allegedly comes up as a mutagen when assayed using the Ames Test (and google for it yourself), it's got quite the reputation for being one of the worst things in the mol biol lab. This is an ideal situation for the talentless hacks marketeers, because it means they can play on lab rats' fears to sell over-priced, under-performing crap.

It turns out that the whole argument against EtBr is so much elephant poo anyway. Perhaps the strongest evidence for the continued use of EtBr is that it has been used for years to treat sleeping sickness in African cattle. And if you do the sums, to get the EtBr dose equivalent that is used to treat one cow, you'd need to drink fifty thousand litres of gel staining solution.

Moreover, when a company decided to test their own EtBr substitute for toxicity and mutagenicity against the 'leading brand', they found that yes, their alternative was safer than the competition, but there was no evidence that it was any safer than EtBr itself:

EBtesting.png

In the graph, notice how SYBR Green — the 'leading brand' — goes up, which is bad, and then goes down, because the test cells are dying, which is very bad. EtBr ('EB') itself seems to be about as dangerous (i.e. not very) as EvaGreen, the company's chemical being tested. Notice that there is no dose-response effect. That is, in general, if something is dangerous, then the more you put into the system the more effect you get. Here, that does not happen for EB (or EvaGreen), which implies that any mutagenicity that is being detected is noise in the system. Indeed, I might suggest that the falling off in 'mutagenicity' in the three right-most EvaGreen columns is itself indicating a cytotoxic effect. Gosh.

And given that these alternatives are generally less sensitive than EtBr, which means you have to use more, EtBr begins to look like a clear winner.

Glory Days

5 September, 2007

Progress is not always beneficial.

Back in my school days, in chemistry classes we used to pipette various liquids by mouth, and think nothing of it. Mouth-pipetting is great; if you're reasonably competent you just grab a pipette, suck, hold pressure with tip of tongue, blow when necessary. No extra equipment required. Of course, this must have been bad for us because school children in their thousands were dying from pipette-transmitted diseases and from ingesting benzine or whatever we happened to be playing with that week.

Yeah.

So, I went to university and found that the Health & Safety cretins officers had decreed that mouth-pipetting was far too dangerous (read, "people might sue us"), as witnessed by the carnage in our classrooms, and that we must use these beasties in our practical classes:
nasty pipette-thing.

These were great: The glass pipettes, when put into the appropriate hole, would easily break, thus enabling cack-handed students to shove jagged glass spears through the palms of their hands (which, unlike the pipette-transmitted diseases and drinking of benzine, I actually observed, first-hand as it were. Twice). Glass pipettes, of course, are far more environmentally-friendly (and cheaper) than the disposable plastic ones that do not break quite so readily, and universities spend too much on Administration (including Health and Safety. . .) to be able to afford a good supply of these fellas:
nice pipette-thing.

I'm saying this because I spent fifteen minutes this morning wandering around the lab looking for one of those bloody things so that I could use a single rainforest-killing ozone-depleting global-warming non-student skewering plastic pipette. Eventually I found one of these
old pipette-thing and was gratified that, after *mumble* years I still remembered how to use one, onehanded. Yay. Go me.

In the old days, I'd have just sucked up the 2xTY and spat it out. Progress, you see?

Oh, and while doing the Google thing to find some pictures with which to illustrate this rambling nonsense, I came across a girl band who call themselves the Pipettes. This tickles me.

a different kind of pipette
(Image credit: foche)

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HeLa cells , n.

hela.jpg

Evil little bastards. I suspect Henrietta wasn't too happy with them, either.

Too much

19 July, 2007

Derek complains that his labs have had too much (redundant) equipment.

Bastard. I wish our lab was so elegantly over-stacked.

Chemicals, on the other hand, oh we have lots of those that have been ordered, used maybe once if at all, and left, forlorn and forgotten, at the bottom of a freezer. And sometimes on the shelf, right next to the marbles:

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Right, I promised to give a not-so-quick list of seminar do's and don'ts. The context for this is the Honours students giving their 'end of labwork' talks in a few weeks, and there's a few scribbles on a Post-It note I'd like to share.

The thing to remember through all this is the point of a talk (or lecture, or seminar, or whatever you want to call it). You are trying to convey a message, and most of the time that message is "Look how brilliant I am". Seriously. You are using your work to convince people in the audience to think "Hey! This cove deserves a degree!" or "Hey! I'd really like this person to work in my lab", or even "I'm glad I employed this person in the first place". Anything that distracts (or detracts) from your message is a bad thing.

That is why I've come down so heavily on the presentation aspect. It does matter, because people (being human) will focus on what they perceive to be wrong, and you do not want them thinking about the bad when they could (should) be focusing on your brilliance. Now, obviously you're not going to please everyone all the time, but you can certainly put your thumb on the scales.

All these points arise from sitting through various seminars. Unfortunately it's not just students who make basic errors; the pros do it too. A couple of weeks ago an email came round the 'academics' mailing list in the Cage, noting that our courses were getting a bad rap in the national press. We've all got something to learn.

The first point I'm going to make is so obvious I'm surprised it needs to be said. But if you get it wrong it's probably the one thing that will make people think "What a prat". It's so important that I'm going to put it on a separate line:

Spell check your slides.

Honestly, this is not difficult (and I'm not talking about technical terms, I'm referring to common words). On my computer at least, questionable words are automatically underlined, but the ability to check spelling in any document has been standard for essentially for ever in computing terms. And if a word looks all right to you but is flagged by your spell checker (Americanisms are a big gotcha, especially because the Americans themselves are deeply confused about -ise, -ize and -yse), then look it up. The OED is free to use from a USyd computer, so make use of it.

Related to this, if you're writing complete sentences rather than bullet points then check the grammar. This is slightly more difficult, but ask someone in the lab to check your slides for you. And if you're dyslexic you should do this anyway (spell checkers will not tell you if 'their' or 'there' is correct in context, for example).

Let's make that more formal. Even if you are an English geek, you should practise your talk in front of a friendly audience before you do it for real, so that they can spot any glaring errors that you have made. Proofreading shows respect for your intended audience. You can work out the implications of that for yourself.

Also remember that certain sad bastards (ahem) delight in looking for words in protein sequences. Find them yourself first; make a joke of it if you like.

Check that your slides are legible, from the back of the room in which you will give your talk. Easy to do, very few do it. Nigel makes a good point from the presentation point of view that serif fonts are generally bad when projected. Times New Roman is just one example of this.

Similarly, the use of colour can make or break a presentation. I still prefer plain black on a plain white background for clarity, with maybe an institutional crest in a corner and a coloured bar (horizontal or vertical) for relief. Recall that 10% of your audience might be colour-blind. Time spent on designing a flashy theme is time better spent on experiments, writing or going to the pub. You are not trying to sell pharmaceuticals to impressionable physicians, you are trying to demonstrate how clever you are. Just because Powerpoint and Keynote have these flashy templates does not mean you have to use them. Remember, you do not want to distract people from your message.

Which leads nicely into the next point: Transitions. I loathe Powerpoint transitions. Most of us have seen them all before and they're boring and, again, distracting. I have to admit that Keynote has one or two nice ones, but they're likely to become just as clichéd in a couple of years' time.

At the opposite end of the spectrum from yellow on blue Comic Sans with whizzing transitions is Sir Paul Nurse. Sir Paul gave a talk a couple of years ago at my institute in Cambridge, and boggled us all by hand-drawing his slides on the fly, on the OHP. Unless you are a very good artist, I do not recommend you try this, at least not until you're as famous as he is.

Keep your slides simple. One message per slide. One, at the most two, graphs or figures and no more than five bullet points — but not on the same slide as your graph! Highlight key points. Do not write out what you are going to say (more on this further down). Try to make all your slides legible without needing to turn down the room lights. This is difficult and not always possible.

The trick is to imagine that Professor Sleepy has dozed off during your talk, and wakes suddenly during a crucial data slide. She should be able to look at your slide and at a glance understand your point. Consider whether you really need to show a sequence alignment. These are notorious to get right, and it's often better just to leave them out. Think about how you are going to show data: It's good to see raw data, but it's also good to see controls versus experimental in a clear, striking graph.

When you finally give the talk for real — and I hate practising talks; I can never get into the swing of it without an audience but as I say, it is essential to get others to proofread your slides and your verbals — there are more things to remember.

Your talk should be structured. There should be a clear introduction; start large and focus in on your speciality. Contextualize. Explain why you did what you did, and what you were trying to find out. Concentrate on the data, and never, ever merely read from the slide. We can see and read that for ourselves; the slides are there to show data and to remind you what to say, to expand upon the core message that you are showing us. Conclude with a clear message.

I don't think it's important whether you give your acknowledgments at the start or the end, or during the talk. When I've described work that a few people have done I like to start by naming and thanking, so that the audience knows who I'm talking about when I say "David suggested we do this". It's possibly better that students save it for the end, but it's not something I'm worked up about.

So you're standing, giving the talk of your life, and you're facing the screen. Wrong, wrong, wrong. When we used glass slides and projectors this was more excusable, but the chances are that you'll have a laptop between you and the audience. So look at the audience, and glance down at the laptop. Ideally you should not even need the slides to give your talk, so except for pointing out things of interest you should never have to look at the screen. Talk to us! If you find yourself unable to remember your slides, and without a screen in front of you, then stand slightly to the side so you can see the screen out of the corner of the eye. Remember you're trying to impress us, not the screen.

Do not rush. Do not whisper, but neither should you shout; project your voice. This takes practice. Use simple terms, not jargon, whenever possible. Pace yourself, and make sure people can understand you. Look directly at members of the audience to confirm that they are getting it (and don't get too depressed if people are asleep. It happens to all of us). Work on your delivery, on capturing and holding people's attention. Consider what you're wearing; again, give no one an opportunity to focus on you or your foibles rather than your message.

"Hold the bloody pointer still", it says here. Most distracting when the little red dot is jerking all over the place while you're talking. Don't fiddle with change in your pocket (my biggest fault!) but don't be afraid, within reason, to use your hands to describe or emphasize. Don't 'um' and 'er', and don't say 'kinda', 'like' or 'sorta'. You may say 'innit' for comic effect. Don't be afraid to use humour, but you had better make sure it really is funny. Don't forget the names of collaborators and colleagues.

At the end of the talk, make it obvious you've finished. If someone asks a question and you can barely hear them, repeat the question (ask them to repeat it if necessary) so that the entire audience can hear. Address the questioner but talk to the rest of the audience as well (a difficult trick. I'm still working on it). If you don't understand the question, ask the questioner to rephrase or repeat, and then repeat your understanding back to them to check (this rephrasing the question in your own words is a useful trick. It lets the rest of the audience hear the question, confirms that you're answering the right question, and gives you time to think). Don't be afraid to stop and take a drink if you're dry; this also gives you more time to think.

I get a real blast out of giving talks, and one of the most important things to remember is to have fun. It's a crucial skill to learn if you're going to make a career out of this game, so you may as well enjoy it. Finally, realize that criticism of your talk is not aimed at you personally. Take criticism constructively, and learn from it.

Ooh, controversy.

I got my Honours student ('W') to perform the experiment yesterday, and this is just in:

freezesqueeze.jpg

The above is a photograph of an agarose gel, which is used to separate different fragments of DNA. It has been stained with ethidium bromide, which glows under ultraviolet light when bound to DNA.

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Into temptation

27 February, 2007

Bull, meet red rag.

donotpress.jpg

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Take me to the river

16 February, 2007

Cage water crisis worsens:

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Strobe Light

14 February, 2007

Heather is whinging about her spectrophotometer. The 'spec' is a machine that shines light of a defined wavelength through a sample and measures how much is absorbed. It returns a quantity known as 'absorbance' or (incorrectly) 'optical density' ('OD'). This is useful to us because the amount of light absorbed is directly proportional to how much stuff there is. Furthermore, different stuff absorbs different wavelengths of light.

So, to pick a completely non-random example, you could make some DNA, put some in a cuvette (the sample vessel, of a defined size) and analyse it in the spec by shining light of wavelengths 260 nanometres (nm) and 280 nm (these are far ultra violet wavelengths) through it. You would find out how much DNA you have, because there is a simple formula that relates absorbance at 260 nm to concentration, and how much contaminating protein there is, because (most) protein absorbs light with a wavelength of 280 nm.

Thus you can therefore measure protein concentration, or follow a colorimetric reaction by observing at the appropriate wavelength, and so on. A spectrophotometer is a very useful instrument, very simple in principle, these days very easy to use, and a source of great frustration to many.

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The hardest part

3 February, 2007

One of these daysyears I am going to get a poster finished a week before the conference.

Let's see: Conference starts on Sunday, my poster is due to be on display Monday afternoon. The preceding Thursday lunchtime I am still doing an experiment and have not started work on the poster. All the rest of Thursday I design and draw a single figure. That leaves Friday to complete the poster, because my children will probably divorce me if I am not at home the Saturday before I disappear to Melbourne for a week.

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Smoke on the water

19 January, 2007

The most important reagent in our trade is water. Most of our reagents are made — and most of our experiments are carried out — in a huge concentration of water. Pure water is 55.5 moles per litre. That's a lot. And if the water that you use for your reagents and experiments is not as pure as it could be, then you could have a problem.

Incidentally, this is why lager in the UK is so crap. When you have a product that is 95% water — because Stella and Grolsch and Heineken and XXXX (because Australians can't spell 'beer', yes I know that one) and the rest of that watery yellow wee, in the UK are not actually imported but brewed under licence — and that water, as safe as it is, tastes dodgy, then you have crap beer. Imported Pilsner Urquell is fine, and locally-brewed ales such as Owd Rodger or Theakston's get around the problem by (a) being brewed where the water is good and (b) having flavour.

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Powerslave

17 January, 2007

Scene just now in the lab: Your humble correspondent setting up an RT-PCR experiment. The conversation went something like this,

Black Queen: Are you going to use the new block ?

BK: No. I have a programme on the Eppendorf.

BQ: But it's intuitive!

BK: You mean you can give your samples to a grad student and let them get on with it?

BQ: <That Look>

Warning Sign

18 December, 2006

The company with the easily accessible information symbols is at it again.

From the (hell, let's name and shame) Fermentas' catalogue entry for Klenow fragment, the fragment of a bacterial DNA polymerase that 'fills in' ends of DNA:

fermental.jpg

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Patent pending

9 November, 2006

Today I have been mostly running gels.

I do a lot of this; it is one of the most useful tools in the molecular/cell biologist's garage. It is used primarily as an identification method (on the basis of relative molecular mass, or more colloquially, size) for DNA and proteins, but often also for purification.

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One of the things that a visiting alien might comment upon, were it to observe laboratory life for more than a few days, is the strange hours that scientists keep. Working longer than the hours stated on the payslip is a badge of honour in academic labs. Calling someone a '9-to-5er' is about the worst insult imaginable.

There are multiple reasons for this. Academic research is competitive, and to compete with the Americans (say) with their huge labs and armies of postdocs we must work longer hours, we are told. Working all day Saturday means you can be nearly 20% more productive than someone who does not. Imagine if your bank account gave you 20% more interest than other banks! Every little bit helps in the battle for publication and funding. If you stay a couple of hours longer in the evening you can repeat that PCR today, which means you will have stolen a day's march on the competition. You can eat lunch at your desk while you write a paper.

Should I go home now, or do that transformation, set up those cultures, spend another hour here? By squeezing extra hours out of each day I can create the impression of industry and expediency, no matter what the real benefit is.

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I walked into the office the other day to grab a notebook, with a pile of shiny, new tissue culture plastics under my arm.

CK accused me of hoarding the flasks and wotnot like a squirrel. I countered by saying that I intended to be the only person capable of carrying out cell culture in a post-apocalyptic world, cornering the market as it were. Think of me as a pre-alcoholic Mel Gibson.

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High Speed

24 October, 2006

Does anyone out there still believe that Pfu Turbo (or Ultra™) is worth it in terms of site-directed mutagenesis? Ricardipus or Hal, any ideas?

Yes, it has been a while since I've done it, but I am certain I used vanilla Pfu last time. Which, for us at least, has certain cost benefits. And I had a go at Hal a couple of months ago on this very subject, it seems.

This imposter is not really a structural biologist; he's one of our IT support trolls testing the 3D capabilities of Coot:

coot.jpg

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Joy in small places

28 September, 2006

Here is the microscope that was used to take those pictures:

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One of my turns

19 September, 2006

One of my fellow Rats commented that she liked doing cell culture, because what with the laminar flow hood and everything it felt like she was doing Real Science. I know what she means, so here's another photograph of lab furniture; the cell culture, or laminar flow, hood.
hoodie.jpg

There is an arrangement of fans and filters with the general idea that any nasty stuff inside the hood stays there and does not infect the operator, and everything on the outside stays there and does not infect whatever it is that the operator is working on. Obviously this theory breaks down a little bit because you have to put your hands and flasks of cells, solutions, pipettes and whatnot inside in order to actually do anything. We get around the problems this causes by liberally spraying 70% ethanol over the surface and anything that gets put inside (including hands, which are usually also washed with Hibiscrub first). There is also a reasonably high intensity ultraviolet lamp inside that gets turned on when the hood is not in use, to make life unpleasant for any nasties that do manage to find a way in.

And yes, you do get to feel like a real scientist doing real science, and most of us doing cell culture form an irrational emotional attachment to the cell line(s) we happen to be working with at the moment. We care for, feed and nuture our cells and it can be quite distressing (not just because of the time wasted) when, not if, you go to the incubator one morning and find your precious cultures swarming with bacteria, or fungi, or strange beings from the Planet Claire.

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Strange brew

18 September, 2006

I want to make up some RNA loading buffer, right? And I'm googling (yes, Virginia, 'google' is a verb) for a recipe, and I find that a certain company not only makes the stuff but publishes the recipe, too. And then I see the advisory symbols for the product:

RNA Loading Dye advisory sign.

Now, I get the '4°' symbol — that means store in the 'fridge. But what the hell does the cup and saucer mean? 'Go and have a nice cup of tea while you run the gel'?

If you have a better idea, please post your suggestions here.

Typecast

15 September, 2006

It's lab talk day today.

And in a quite possibly doomed to utter failure attempt at spreading some reason and clarity in this troubled world, I would like all students to BAN COMIC SANS from their overheads.

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"Dear Dr Fleming,

Your lab is a mess."


Cell biology is a messy science.

Cells never behave how you want or expect. Except when you least expect it.

Some constants: Transfections are variable. Mountant is messy. Immersion oil is messy. Data are never unequivocal.

When will I be famous?

10 September, 2006

1. Working out primers that would distinguish between two splice variants.

notes

2.

(a) A sketch to help me explain something to P
(b) Various 5' splicing sites
(c) The molecular mass of something or other and a random note
(d) A couple of names jotted down after a conversation with my ex-boss when he visited.

When I'm famous I will auction off these memorabilia; or you can make me an offer now and beat the rush.

Epiphany

31 August, 2006

Hal has had a revelation.

Stratagene’s QuikChange Mutagenesis Kit:

Contains: PfuUltra™ High Fidelity DNA polymerase, 10X reaction buffer, dNTP mix, Dpn I restriction enzyme, QuikChange control plasmid and control primers, XL-1 supercompetent cells, pUC18 control plasmid.

I'm wondering what took him so long. I first used that method in, oh, 1996, 1997? Stratagene (patent filed in 1995) had brought out the kit, and I looked at the method and thought what is stopping me doing that with standalone reagents?

So I talked to my then-boss, ordered the kit and the individual reagents from different suppliers, and did the side-by-side comparison. Since then, I think I've ordered the kit precisely once and that was to get hold of the instruction manual, which is now available online. And made countless mutants with DpnI from NEB and bog-standard Pfu.

You can even use Taq with high fidelity if you use dNTPs near the Km . . .

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Slap one on

30 August, 2006

There's an interesting phenomenon that occurs in labs.

In normal life we find it convenient to label things. It helps us organize our lives, recognize authority, and in some cases limit concepts and people so that we can deal with them; Takei is Japanese, Samantha is an arts graduate, Larvatus Prodeo is a whiny leftist blog. There are labels that, even with the best intentions, demean the very thing that is being labelled but in a way that constrains it sufficiently for us to understand, cope, deal with it.

And more prosaically we label the sugar and the salt so that our pavs lov.

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. . . But my heart's not in it.

It is quite a change for me to be in a university setting. The Council for Biomedical Research, although avowedly academic in nature, actually had very little to do with the local tertiary education institute. We had on average one graduate student per group, their stipends usually funded by the Council's fiercely contested fellowships. Local students were not advantaged, and the only time we got to see an undergraduate was when one was poached for the summer, via personal contacts.

So being in a full-on teaching department is a bit of a culture shock. I'm surrounded by students. It's not a problem, it is just different. Being old enough (legally, even) to be the father of some of them has not freaked me out; in fact I find it quite invigorating. Not just in a 'I shall mould these youthful, malleable minds to do my bidding mwah hah hah ahem' way, but more in a 'gosh these guys are keen' sense.

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Friday wibblings

11 August, 2006

I am sure that most people in my lab could pass as perfectly normal members of society. I know that some of them even have real lives, and seem to be able to maintain relationships outside the lab.

Therefore I have been somewhat taken aback by the response to the Axygen 'eppendorf' tubes. A fair proportion of the lab rats have come up to me in the last week and raved - there is no other word for it - about these tubes. All right, they are good, but I never expected such an emotional excess.

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Tame reps

10 August, 2006

Hal's post reminds me: I bumped into Dan from PLP (see this post) in the corridor yesterday. Apparently the poor bloke had been losing sleep because he thought he had promised to bring in some more samples and could not remember bringing them, or indeed what they were.

Curse my honest nature; I failed to seize the opportunity to turn a fast buck and told him I had everything he had promised. But this customer-focus is exactly the kind of thing that means I'm more inclined to consider PLP for other products. They don't do culture plasticware but I did managed to extract a 96-well PCR plate from him.

Which reminds me, as our thermal cycler is broken I had better run and book the other lab's one . . .

You tube

26 July, 2006

No, not the video place that is taking teh intaweb by storm.

'Eppendorf' tubes. You may remember I was wibbling about sub-standard tools of the trade. The Greiner rep did indeed show, and was appropriately chastened. I got a free replacement box of 8-tube strips (which appear to be all right) and a sample of eppendorf tubes made by Molecular BioProducts.

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True life

17 July, 2006

After last week's little rant about the portrayal of science in the meeja, I thought it might be interesting to show you a couple of photographs.

This blacklab.jpg is the view from the lab door.

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I revisited the gel-eating tube question yesterday. I did the experiment in the suspect tube strips and in another brand in parallel.

Guess what? The other brand's reactions were fine, the original strips again ate the gel. How's that for science in action?

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Mycoplasma

12 July, 2006

The word alone chills the hearts of experienced cell biologists. And when, a couple of months back, someone upstairs was getting strange results from their cell-based assays, the Boss came to me and asked,
'What do you know about mycoplasma?'. The icy black hand of dread gripped me and I spent the rest of the day trying to find a supplier for a mycoplasma detection kit in Australia.

I spent the next two days arguing with various suppliers and AQIS about what I could or could not import and for how much. It seemed that AQIS were upset about the presence of mycoplasma DNA; in effect the small amount of positive control included in the kits I was looking at.

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About the Rat

Black Knight is interested in the interaction of science (as a day job and as a way of thinking) with his family, the wider community and literature. And tormenting students. Frequently polemical, sometimes serious, and hopefully always entertaining more

blackasknight@gmail.com