a worthy competitor

Due to the lack of other fourth-year PhD students to compete with, my lab mate and I have decided to compete with phd comics' Cecilia, who is also thinking about writing up her thesis. Given that Cecilia is an American (and we all know how long PhDs in the USA take) we're going to be really gutted if we lose to her (especially since she's imaginary).

In addition, my lab-mate and I are competing against each other. Whoever loses buys the other a pint. It would be buying each other a pub dinner, but as we have no job lined up as yet, and our funding has run out, we decided to make it a more economical prize.

Hopefully we won't die of thirst first.

the next generation

I just found out that my oldest friend is pregnant! And not just pregnant, but 8 months pregnant! How come it's been so long since I last talked to her? We have communicated by sms, but that bit of news was omitted....

Wow. I still remember having sleepovers at her house with the clown nightlight in her room so it wouldn't be completely dark - and being more scared of the clown when I needed to get up in the middle of the night. I babysat her tamagochi and her guinea-pig when she went on holiday to spain. I remember being mildly grossed out when we found out she was going to have a little brother (we were ten years old by then and thought our parents were ancient). I remember when that little brother was in a cot. And now she has a cot in her room for her own baby.

She says it's payback time for her brother - after all those years of babysitting him, he has to babysit for her now. He's only 14 though, so it might be another 2 years before he's legally allowed to babysit for her on his own.

I have other friends who have had babies, and the number of children belonging to my generation of my family seems to grow at an alarming rate. But somehow this is harder to take in. Perhaps it's because I only have a month to get used to the idea before the bump turns into an actual baby, or perhaps it's because I remember being seven years old with my friend. And the screams she made when we cycled into a (parked) car and scraped her knee. She didn't scream when she actually scraped it - she screamed because the antiseptic her mum put on stung so much.

But I think I'm looking forward to being back in manchester and saying hi to the new arrival. Perhaps I can teach him/her all the naughty tricks we did then?

the last of the fourth year students

Aaargh
I'm officially the last one of my batch of phd students not to have submitted their thesis!! And I haven't even left the lab yet.
I'm hovering between panicking and complacency now that I'm the last one - I can't be any more last than last. Except I know that people who started after me are also starting to write up.

Oh and yes. I finally set up my cantab address. Not that I'll be leaving anytime soon.

Wallace and Gromit rise again

I watched Nick Parks newest Wallace and Gromit short last night - A Matter of Loaf and Death (cue appropriately dramatic and scary music).I love Wallace and Gromit, the writers have a brilliantly wry and British sense of humour - and as my friend points out, they actually create a storyline which works without taking itself too seriously. The amount of attention to detail and the painstaking effort required to film it frame by frame is worthy of our appreciation in itself. How is it that they can convey more expression via modelling clay than most computer-generated animations seem able to do?(excepting some Disney and/or Pixar films - I won't concede all.)
The best bit for me is the waking up of Wallace scene, with a *new and improved* bakery-incorporated method of getting Wallace out of bed and into his trousers (literally). My glee amplified when the contraption played an extra starring role later in the clip.
A bit floury, very nutty and very well done.

What should have happened...

This is what I wanted to achieve with my laser capture microdissection work. This is the cross-section of a tobacco leaf. I've used the laser to remove only the upper epidermal cell layer (The section is upside down). I had to camp out for a week in a room cooled to 12 degrees Celsius, with the cooling motors making a racket in the background, to get these tissue sections.

shooting practice

I spent a very frustrating couple of hours on the laser capture microdissection machine on Saturday. The aim was to capture a sample of about 250 cells from a fixed leaf section of 5 microns thick and extract the RNA from them, to use for RNA analysis. The basic idea is very simple - you have a 'cap' of thin plastic film which you direct the machine to position above your tissue section. You can then look at a microscope image which is projected onto your computer screen and mark which individual cells you want to capture. The computer then relays this information to the laser arm which fires a laser beam for a period in the microsecond region at the cap above each point. The film then melts, adhering the cell as it reforms, so that when you lift the cap off the desired cells should be adhered to the film.

Very cool, and I've had brilliant results from it. The hardest part is only really optimising the laser parameters, as it has to be strong enough to melt the film, but not so strong that it burns a hole in your tissue. However, it seemed that the laser just didn't want to play on Saturday. That or it was too eager. It jostled between not melting the film, only making dents in the surface (see top image) or burning a black hole in the middle of the target point. As the RNA in the tissue section only remains intact for an hour or so after wax deparaffinization, by the time I had the laser optimised there was no time to capture the cells I wanted.

Not yet defeated by this failure, I prepared a second section, and set about re-optimising the laser for this sample (you have to re-set everything each time you change samples). Again it wasn't playing. My cap looked like it'd been used for target practice.

In the end I gave up. It was only when I was removing my caps from the machine and complaining to a friend that he noticed a dust-hair on the surface of the cap. Those very fine ones that float about. But it was enough to prevent the cap from sitting flush against the tissue surface - hence my problems with the laser. Sigh. At least now I know, so I'll be cleaning the cap holder very well before using it in the future.

spoke too soon

It turns out that the end of the light of the tunnel was really just the headlights of an on-coming express train.

I've been trying to put together the figures for my *first* paper (yay). It's a mix of my work and some results from a PhD student who finished last year. My supervisor's writing the main bulk of the paper, which is good because it saves me time, but he's assumed we have images and figures that we don't have, which is BAD. We have the data and some of the photos, but they were meant for our own records and not of publishable quality. The other problem is that the previous student's thesis is a nightmare to trawl through, there are so many different plant lines and he's laid out the results for each experiment in a slightly different format each time (why oh why?!!!) .

Some of these issues I need to bring up with my supervisor, but in true traditional supervisor fashion, he sent me the rough draft on monday and then disappeared for the week on tuesday. I need to give him a mock-up of the figures at the start of next week, and there's been no word from the previous student in response to my cry for help (and data in a more sensible form).

At least I'm lined up to be joint first author on this paper, rather than the second author I thought I might be. But it's a hefty amount of work I need to do to get it.

a single keystroke

I need to start writing up in earnest.

The end is in sight

Over the last few days, I've thrown out 7 shelves of plants - that's 45 trays or 315 tobacco plants. I did take samples from them, so they didn't die in vain. More to the point, I'm in the last stages of processing my data from over 300 samples.
I've also heard that each growth chamber at the plant growth facilities cost us £4000 a month to maintain. As I've been using half a chamber, that's £2000 a month I've been clocking up for the lab bill, if the rumours are true that is. Our group alone use the better part of 5 rooms, adding up to a ridiculous amount of money spent on lighting and humidity control alone...

Anyway, hopefully not too long left doing experiments!

Plants versus zombies

It's amazing what we can do with G.M. technology these days.

Pregnancy tests for viruses

There are several ways to check if your plant is infected with Tobacco mosaic virus (TMV). If your plant of interest is Nicotiana benthamiana (i.e. such a sickly thing that it wilts at the mere sight of a virus) then you wait for them to die. Though that could mean it was infected with any one of scores of plant viruses.

To narrow down the list of culprits, and to do so before plant death, then look out for the dark green veins surrounding light green patches of leaf tissue, where chlorosis has occured due to insertion of the virus coat protein into the chloroplastic envelope. The leaves will become mottled and curl up. These symptoms could be attributed to a much smaller number (i.e. less than 10) of different virus species that cause the same symptoms.

If you're more pedantic about your viruses and want to know if your plant has TMV, you could use antibodies against TMV-specific proteins, or primer sequences that complement TMV-specific RNA sequences in molecular-based assays.

However, you can also get tests to check if your virus is TMV/CMV/boy/girl/indeterminate gender. They look like a pregnancy test, work in the same way as a pregnancy test, you interpret your results in (I'm told) the same way as you would a pregnancy test. The only difference is that instead of peeing on a stick you put plant extract on it. (And no, you cannot train plants to pee).
And yes, my brain is addled from too much data analysis.

M.I.A. - Have you seen this PhD homo sapiens?

I haven't updated my blog for a LONG time. I've found that being an unfunded fourth year PhD is a time-consuming job, despite being technically unemployed. Not only have I become jaded and bitter, but my life is increasingly asocial and self-centred. My thoughts are unable to drag themselves away from the *exciting* lab experiments that pre-occupy them (and inevitably don't work) long enough to compose any meaningful or entertaining prose. Of course, that has always been the case, so what's new?

Anyway, I realised that this lack of communication with the world outside my lab room 123 was getting out of hand when my mum had to resort to sending me a facebook message to check I'm still alive. I shall endeavour to be something more resembling a human being.

Please humour me if my next few blog updates are all work-related. It's an uphill endeavour.