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Saturday, April 02, 2005

Desperately Seeking Data (Part 1)

Well, its that time of the year again - time for our annual conference. Sadly, it won't be as glamourous as Jonny B's recent jolly to Rome (as a non-delegate, no less), rather a national conference daan saarf (as Southern Bird would say), but we usually put on a good show with plenty of free booze and a couple of decent feeds. I'll also get my annual greasy fried breakfast as well.

Of course now that I am a PostDoc and have been doing this project for some time, I am expected to present some results. Hah! This year it was decided that I would present the "molecular" side of my work, so I duly submitted a 200 word abstract 2 months ago promising the results of the experiments that I was performing. Yes, I really did think that I would have some results from my ongoing, Tourettes-inducing, Southern blots.


Three weeks ago I received an email from the conference organisers "Congratulations, Dr SaneScientist, you have been selected to present a talk based on your work".

Double shit!

At conferences, delegates are invited to submit abstracts detailing their work. The abstracts are then discussed and those that fit nicely into the themes that the conference organisers wish to discuss, are selected to present a short talk. Each slot is 15 minutes, split into 10-12 minutes of snazzy Powerpoint slides followed by 3-5 minutes grilling/questions by the audience.

The other abstracts are instead offered a Poster slot. The author prints out a brightly coloured A0 poster which they stand beside and discuss with other delegates, whilst consuming coffee and biscuits.

There's no question that it is a small honour to be given a talk rather than a poster, but frankly, I don't have nearly enough to talk about. I was expecting a poster. If you make your pictures big enough you can put surprisingly little data on a poster. And if you are really worried about your work, you can just bugger off, cover your name badge and avoid anyone who looks like they may have a question. It's a little more difficult to bullshit and waffle for 10 minutes in a crowded lecture theatre full of your peers whilst standing on stage with a radio mike.

Oooooh shiiiittt.......

So it was decided that I would present a few key points from a former graduate student on the project then discuss in detail the results from my Southern blot data. Those experiments were expected to A) Work and B) Yield fascinating data that will grip the audience and have them clamouring for more. Or at least lift their heads of the desk, ignore the hangover from the previous night and attempt to bring my slideshow into focus.

So I increased my workload. Significantly.

At every turn, however Sod's law kicked me in the bollocks.

First there was the palaver with the wrong PCR primers being sent to me (See the MWG-Biotech Tuesday Twat award for more details). That wasted a week. A hasty revamping of my timetables showed that I could still perform the three blots that I wanted to do in 2 weeks. The early Easter meant that I had to miss a weekend, but I could cut short my holiday. The weekend before Easter, I had arranged to go see a mate in a play in London. No getting out of that, so yet another 3 days out of the lab. By careful timing and accepting the need to work 14 hours a day I figured I could compress each 5 day procedure into 3 days.

I did my first blot. It failed. The image on the computer screen should have been a series of crisp black bands highlighing the gene I was looking for. What I actually got, was an indisinct black smear, showing absolutely fuck all information of any use. Shit. 10 days until the conference and Easter weekend coming up. And what was worse, I had used up all of my remaining DNA stocks.

Arse. Ok, still possible. I could grow my yeast cells up and extract some more DNA, then perform the first blot, then the 2 remaining blots at the same time the week after easter, just stagger them by a day. So I innoculated a batch of cells and left them overnight to grow, ready for DNA extraction in the morning.

The following morning I opened my flasks, expecting to smell the faint beery waft of fermenting yeast. Nope, a faint musty smell. A quick look under the microscope confirmed my fears - bacterial contamination. Fuck! Bacteria reproduce about 5 times as fast as my yeast. In an overnight culture, the number of yeast cells doubles roughly every 2 hours. In the same period of time the bacteria will have increased by a factor of 32. Over about 24 hours, the number of yeast cells will have increased by about 100,000 times - these bacteria will have increased by several billion times! Therefore, even a few stray cells will massively overwhelm my yeast population. Worse, the source of contamination was my stock agar plate, probably contaminated by me or my colleagues' coughing and sneezing the previous week.

Shit! What to do? All I could do was try and cure the contamination. A colleague identified the species of bacterium and recommended an appropriate antibiotic. I smeared a series of agar plates with liberal amounts of the antibiotic. I then took what I hoped was a relatively uncontaminated part of the stock plate and smeared a miniscule amount of these cells onto my new plates. It then took 2 agonising days to grow. It was now the morning of Good Friday, and my train ticket was booked for lunchtime. The agar plates looked OK - but then this bacterium looks like yeast to the naked eye - so no I couldn't be sure. The plate at least smelled like a wino's armpit, so that was good. I had found a colleague who was going to be working over the easter weekend, so I bribed her with chocolate to take my culture out of the incubator when it was ready and set up a new culture (with shit loads of antibiotic just in case) and went back to Mum and Dad's for Easter.

A look at the train timetable revealed that if I cut short my Easter vacation and travelled back on the bank holiday monday morning I could be back in the lab by lunchtime. I cancelled lunch with an old school friend, promising to discuss her upcoming nuptial's over the phone, and came back to work. The culture had grown! And it smelt of beer! WooHoo! By midnight I had extracted just enough DNA for one attempt (you can guess where this is going...).

Next step was to perform the first steps of the Southern blot. I calculated that if I restriction-digested the DNA overnight (a fancy term for cutting the DNA into different-sized chunks using a specific enzyme), then ran it on a gel early the next morning for about 14 hours, I could start my southern blot that evening and be out of the lab by 4am. I would get the results for at least one of my blots before my next "progress" meeting with SWMNBN on Thursday and have enough time to insert it into my talk for Friday morning's practise in front of the rest of my lab.

I opened my freezer to look for the restriction enzyme that I needed to use. The previous week, about half a dozen different enzymes had arrived for me when I was out and someone had placed them in my freezer. I found every single enzyme I'd ordered, all sealed in their boxes - except for the bugger I needed. WTF? The order form confirmed that it had arrived and been signed for. Shit, where was it? 20 minutes panicing later and I found it unwrapped, sans instruction insert in the communal freezer. The lack of an instruction insert wasn't a problem. The hundreds of different enzymes available from suppliers typically work best diluted in 1 of about 8 different buffers, however the coloured dot on the lid of the enzyme's tube corresponds to the dot on the buffer tube. You simply dilute your DNA in water and the appropriate buffer, add the enzyme, stick it in a water bath at 37C and leave it overnight.

No problem....

The next day I came in early, took my DNA, and loaded it onto a gel to separate out the digested DNA according to its length. I then started preparing the DNA for my second Southern blot, to be started the following day. By the late evening I was already tired, but ready to continue with the first blot. I photographed my gel under UV.

FUCKITY FUCK FUCK! The DNA hadn't digested! Instead of a faint smear I had a bright, single band of undigested DNA. Shitty shit shit! Why? What went wrong? I double-checked the enzyme tube. White dot. I opened the buffer box, white dot = Buffer L. Yup, definately done that. And I added an excess of enzyme so that wasn't the problem. In desperation I logged onto a restriction enzyme database and looked up the enzyme's instruction sheet.
10 units per microlitre concentration. Check.
Digests at 37C. Check.
Requires Buffer L. Check.
I read down to the small print. "Also requires addition of 1xBSA for optimum performance".
Bollocks! I couldn't believe it! The one time somebody goofs and throws away the intruction pamphlet, the enzyme is one of the handful (out of hundreds) that requires something else in addition to the standard buffer.

I have decided that it would be best not to ask who picked the enzyme up. Those night owls still working who heard my expletive-littered rant will no doubt have let it quietly be known that SaneScientist would in future appreciate it if boxes labelled "SaneScientist" are delivered intact to his freezer, rather than unpacked, picked apart and buried in the communal freezer.

So at this point (Tuesday night) 2 days remain before my next progress meeting. 3 days until I run through my (as yet unwritten and barely planned) talk in front of the lab. 5 days (and yes I count weekends) before I hop on a plane to the conference.

Stay tuned for the next thrilling installment, when you'll also find out why I had the time to write this lengthy blog entry on Saturday night...



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