I wrote the article below for the University of Manchester, where I'm pursuing a PhD in Plant Sciences. There's some real science, as well as comedy gold in this article - though it's not necessarily for the lay person, it does describe a lot of the basic procedures that you need to know in molecular biology!
8am - 10am ¦ PlanningI awoke to a gloriously sunny day in Manchester, albeit slightly groggy due to a late night previously. I had been to see John Bishop in Blackburn and drove back at around midnight! Waking up is always difficult, but I’m better at working in the mornings so I got into labs at about 9.15 (not before I bought my usual bacon butty and regular latte from Greggs on the walk there). The walk wasn’t all plain sailing however, as the first signs of frost nearly caused me to stack it outside the local primary school. Thankfully my blushes were spared by a fortunately-placed lamp post... Between my arrival and 10 o clock I organised the week ahead by looking through my last week’s work and seeing if there were any outstanding experiments that needed to be done.
10am - 12pm ¦ LabAt 10 I entered the lab and started a ligation reaction (for the third, and hopefully last, time) as my initial experiments for my project involve creating vectors for artificial micro-RNA (amiRNA) knockdown of three CAX genes in the green alga Chlamydomonas. In order to maximise the chances of one of my transformations working I have been using multiple vectors and multiple inserts – though some have proved more difficult to combine than others! Fingers crossed by the end of the week I’ll have a full set (for CAX1 at least...)! I also started a sequencing reaction for another of the vector + insert combinations – using coloured PCR tubes... the colours make the experiment seem less tedious!!
12pm - 2pm ¦ LunchThe hour between 12 and 1 was spent hunting for food with my pal Matt who is doing an MSc in Plant Science and did the same degree as me. After trying the Oxford (closed), then Gemini Cafe (teeming to the brim), we decided on some food at University Place. I had meatballs and pasta, which was half-decent – though I miss the roast dinner sandwiches from the old Refectory.
2pm - 5pm ¦ LabI retrieved the sequencing reaction from the PCR machine and performed a clean-up reaction. The smell of sodium acetate was actually quite alluring, though maybe that’s because it masked the putrid stench wafting from the autoclave bin near my feet... Still - not long after that ethanol had to be added which made me dream of going to the pub later on. The view from the lab window was particularly pleasing today as well, so I took a photo. The view from our lab is quite good because we’re situated on the third floor – on a nice day downtown Manchester is quite prominently in view!
5pm - 6pm ¦ FootballI played a 6-a-side football game for my team “Saturday Night Beaver” between 5.10 and 5.50. I lost count of the score in the end as it wasn’t a good day at the office! Although we did concede half the amount of goals in the second half than we did in the first half, which is an improvement if nothing else. It’s also the fun that counts (and hacking down the opposition).
7pm - on ¦ PubAfter a resounding defeat the pub was the only option. We went to the Red Lion in Withington as it’s a proper pub that serves Ale and such. At first there were 4 of us, but by 10pm there were 12, which made for some good craic all round. Highlights of the night included my friend Jack being accosted by a much older lady, who came out with such classic quotes as “You’re a dark horse aren’t you?!” etc... Another laugh was bumping into the Manchester University Jazz Orchestra - there were plenty of chances to slip in jokes, mostly regarding big instruments. We decided to walk the 40 minutes back to Rusholme down Wellington Road, helped along against the cold by the buy-one-get-one-free hot and spicy pizzas that we purchased from Khans.
8am - 10am ¦ TravelAnother fresh day in Manchester with the sun shining made for another enjoyable walk to the Smith building (via Greggs again). Upon arrival at university I walked over to the plant growth rooms to collect some algae from the shakers in order to inoculate a new culture prior to transformation. The plant growth rooms are always a good place to go as they’re full of different plants under a variety of conditions for everyone’s experiments in the department. Unfortunately we’re all supposed to wear some horrible green labcoats in the growth rooms now (see photo) but they look too much like hospital green for my liking so I’ll stick to white! On the walk back from Stopford noticed the church looked good in the sunlight so snapped another photo...
10am - 12pm ¦ LabI wanted to collect lots of plasmid DNA prior to transformation of my algae therefore I performed a DNA MIDIprep on bacterial cultures that had been transformed with the vector containing my amiRNA insert. Before the MIDIprep, however, I had to inoculate LB media with some of each colony and leave overnight in a 37deg shaker. In the meantime I transformed some competent DH5α E. coli cells with my ligated vector from yesterday – third time lucky for this combination!! I then wandered downstairs to chat to some of the other students down there, as it’s always useful to chat about each other’s projects... other people’s ideas can be very useful if you’re a little stuck sometimes!
12pm - 2pm ¦ LunchI read some New Scientist for a break before going to MacDonalds for dinner with Nick (from downstairs) and my mate Jack. A trip to MacDonalds is always more gluttonous with a student card because you can get a free cheeseburger or McFlurry with any large meal, so I did. I always eat my meals on the ledge running alongside the Stopford building, which is nicely bathed in sunlight whenever it’s not raining (a rarity in Manchester, yes). It being November I definitely regretted ordering the McFlurry instead of the cheeseburger, but never mind.
2pm - 5pm ¦ LabI retrieved the E. coli from the 37 degree shaker in the lab downstairs and plated the (hopefully) transformed cells onto a selective LB medium containing Ampicillin. For the rest of the afternoon I did plenty of reading up for the looming literature review. In fact, in reality I spent the best part of 3 hours typing various combinations of “Plant, calcium, signalling, pH and Chlamydomonas” into Web of Science. This can be quite disheartening – especially when you’re scanning through page 30 of the results to try and find a paper that is remotely relevant to the subject you’ve typed into the search boxes. So much for “advanced search”. I’ve decided it’s much simpler to find a review paper on the subject at hand, then use their references and start a “paper trail”. This works exceptionally well until you want a 2010 paper, though Google Scholar
is always useful for that.
5pm - 6pm ¦ HomeI went home at 5, made a brew and watched “The Trip” on iPlayer.
7pm - on ¦ PubI spent the evening with my mate Tim who has just started his first year at Uni after doing his National Service with the Singaporean army for two and a half years. We went to the Oxford pub with all of his flatmates from halls to do the pub quiz. The quiz was fun, although out of 27 teams we would have come higher than fourth place if we’d known more about the Crimean War and naming Scottish castles. Still, fourth place wasn’t bad – though all I had to show for it was the loss of a tenner on three pints of Guinness! We were also robbed in the art round – the team on the table next to us had listened in on our idea and produced a much more detailed picture of it... harsh!
8am - 10am ¦ TravelI arose to a particularly disgusting day in Manchester... in fact the weather would have made the perfect “pathetic fallacy” for a video for the Madness song “Grey Day”. Still, I had the usual bacon butty and regular latte to cheer me up and arrived at Uni for 9 30. It was a much more relaxed day than this time last week when some friends and I journeyed down to London to take part in the HE cut protests outside Parliament. The scenes at Millbank were tame at most and exaggerated by the media – though the drum & bass and fires were fun to stand around with a few cans of beer. We also got interviewed on Japanese TV, result!
10am - 12pm ¦ LabFirst of all I retrieved my plate of transformed bacteria from the 37 degree incubator – which was particularly smelly today. In fact, one of the downsides to molecular genetics work is the smelliness of the organisms you have to work with. Thankfully, Chlamydomonas isn’t that smelly – just your average alga, but Agrobacterium – a bacteria that is used to transform plants through engineering it’s tumor-inducing T-vector – is incredibly smelly and can be quite disgusting to work with. In addition to that, Mayo – another PhD student in my lab from Nigeria – is using sewage in his algae experiments. I fear all of our pipettes are now contaminated with things I’d rather not think about.
I used 8 of the colonies on the retrieved plate to perform a colony PCR to check for the presence of my amiRNA insert and then streaked each of these onto a reference plate which I left in the fridge for future use. E. coli is amazing and can survive dormant in a fridge for absolutely ages... I never fail to be impressed by the species.
12pm - 2pm ¦ LabI performed a DNA MIDI-prep on the 100ml cultures of E. coli that I had prepared yesterday that were known to contain my vector and insert in the correct orientation. The MIDI-prep ensures that there is plenty of DNA available prior to transforming my Chlamydomonas cells. Thankfully there is a kit available for this which is incredibly easy to use. Once I’d finished the prep I mixed the eluted solution with isopropanol and centrifuged for an hour to precipitate the DNA. While the centrifugation was taking place, I retrieved my samples from the PCR machine, loaded them onto a gel and performed electrophoresis to check the band size of my products. Juggling experiments is a pretty common feature of lab life! Electrophoresis is always pleasing because you get a nice visual result in the form of DNA bands glowing in
response to UV light.
2pm - 4pm ¦ LabAfter some food I checked my gel, which (amazingly) showed two bands for a construct that had, until now, proved rather difficult to obtain. It’s these little victories in the lab that make everything seem worthwhile, even when nothing seems to work at first. I then cleaned up my precipitated DNA using ethanol, which signalled the end of the day’s experiments.
4pm - 6pm ¦ Literature reviewThe late afternoon was again spent doing my literature review, as well as checking the results of the sequencing reactions from earlier in the week. Unfortunately, as per usual, the reaction yielded barely any results. I’ve decided this can’t be my fault as previous reactions have had quite haphazard results, so our lab decided to use an external sequencing service from now on. They do the sequencing reaction for you as long as you provide the DNA and primers, which is fine by me as that reaction has taken up a lot of my time in the lab this semester! Just before I left Uni I set up a restriction digest reaction in order to linearise my vector prior to transformation.
Linearised DNA is more easily incorporated into Chlamydomonas genomic DNA than plasmid DNA, for obvious reasons.
The reaction was left in a 37 degree water bath overnight in order that all of the DNA was digested.
4pm - 6pm ¦ RelaxAfter Uni I visited friends in Withington and went for a Chinese – I couldn’t resist the smell of the takeaway underneath their flat. That flat is also the home of Esteban the mini black cat, who deserved a photo. Today was Eid, so a few of us went for shisha and ice cream at Arabesque cafe. The apple shisha was sublime as per usual, while the mint shisha is definitely a new favourite – and went particularly well with the huge chocolate and nut ice cream I had, which looked suspiciously like vienetta... One peculiar revelation from the night was that coffee-flavoured shisha tastes like mackerel. I’d advise anyone to never try coffee-flavoured shisha. Eugh. On a more positive note, the Arabesque Special Smoothie was delectably tasty and I left feeling pretty full (after two).
8am - 10am ¦ TravelI arose at eight in order to get to Uni early, only to realise that my student card was missing. I’d like to say this was a rarity but I was forced to go to student services and cough up a tenner for a replacement card for the third year in a row. I really need a wallet. I went to Greggs again, but mixed it up a little by getting sausages on the bacon butty – living the dream.
10am - 12pm ¦ LabI plunged my restriction digest into a 65 degree water bath for 20 minutes to inactive the restriction enzyme, then ran the product on a gel to purify the DNA and to check that the digestion had worked successfully (it had). I then had to extract the DNA from my gel prior to transforming my Chlamydomonas using another kit (amazingly handy).
12pm - 2pm ¦ LabUsing my purified, digested vector I transformed my Chlamydomonas cells. The glass-bead method of transformation is possibly my favourite as it is so ridiculously crude. The method basically involves mixing cell wall-deficient algae with the DNA vector, glass beads, polyethylene glycol (PEG) and boiled salmon sperm DNA. The glass beads provide a greater surface area for DNA to be taken up by the algae, while the transformation is also mediated by polyethylene glycol (PEG). The salmon sperm DNA is there to encourage the cells to take up DNA, of any sort. The mixture is vortexed for 15 seconds, then plated onto selective medium and left to incubate in bright light for three weeks. Simple! It’s a shame I can’t get the technique down properly yet, as I still have no transformants. Oh well.
It may seem like this took a long time, but unfortunately the algal growth room is in the Stopford building, which involves the long walk via the bridge link and back. Still, that walk is usually good for thinking through the next experiment of the day!
2pm - 3pm ¦ LunchI met a mate from school for lunch at Big Hands. He is now doing Medicine at Manchester and intercalating in physiology. We had a good chat about the events of Monday night – he’s one of the members of MUJO – the Manchester University Jazz Orchestra. Another interesting revelation – feta cheese and chorizo go together amazingly well, helped down with a pint of Tuborg... The world’s smallest ever flower shop is also next door to big hands. I bought a couple of succulents from there at the start of the semester, as well as a plant with pink veins to decorate my house. As a plant scientist I should probably know what they’re called, but you can’t know everything. I’m more of a molecular geneticist than a taxonomist anyway! Or that’s my excuse.
3pm - 5pm ¦ Literature reviewFrom four onwards I decided to do yet more reading for my literature review.
5pm - on ¦ RelaxI went home at 5 and decided to have a quiet night in, considering the week had already proved rather expensive after the pub on Monday, quiz Tuesday and shisha/ice cream/chinese on Wednesday. I also had some homework from my French lessons to do, so I read chapters 3 to 6 of “Le Petit Prince” – a French fairytale. I also had to answer various comprehension questions regarding the text, though the story was bizarre and would have been hard to analyse in English never-mind French. I tried, however, and came to the conclusion that the mystery visitor in the book was an alien from an asteroid who was concerned with a plant infestation on his home planet. As I said – bizarre, yet oddly topical what with the invasive Japanese Knotweed, Himalayan Balsam and Rosebay Willowherb dominating our wastelands, unkempt
gardens and riverbanks at the moment.
8am - 10am ¦ TravelThis was another splendid day in Manchester therefore I walked to the Smith building through Whitworth Park. I wore white trainers though, which was an error. The tramp who usually sleeps under the big tree at the entrance was also missing, probably because the council had decided to waste tens of thousands of pounds of public money on a perimeter fence that could only keep out the smallest of people (including the tramp who is tiny, maybe that’s why they built it - he does smell strongly of urine).
10am - 12pm ¦ Supervisor meeting / labAt 10 I had a meeting with my supervisor to discuss how my experiments had been going that week and to see where I could take my experiments next week. These meetings are always useful as my supervisor always highlights good papers to read for use in both my literature review and my experiments. I then went to the Stopford building to collect another strain of Chlamydomonas, this time both cell wall and arginine-synthesis deficient so that I could doubly transform cells at a later date. I performed another transformation reaction and left plated mixtures in the incubator. On a random note – the arginine-deficient strain was a lovely blue-green colour. Just before midday I performed another sequencing reaction using DNA from the failed reaction on Wednesday, using a different primer just in case that’s what the problem was.
I wasn’t holding my breath, however.
Between 1 and 2 I performed another DNA MIDI-prep on yet more transformed bacterial cultures. The more combinations of vector and construct the better – as successful transformation can be a numbers game if anything.
2pm - 5pm ¦ Literature reviewThe afternoon was again occupied mainly by reading for the literature review - I won’t add any more, same old.
5pm - on ¦ Pub / night outAt the end of the day on a Friday I’d normally go to the “Bowling Green” for drinks with the lecturers and postdocs in the Plant Science department, but earlier in the day we’d bumped into a mate from 3rd year plant science who is now doing a PGCE who told us to meet him at the Student’s Union at 5. We obliged, and sank a good four or five beers while chatting to various teachers-to-be in a very busy SU. It’s now £1.50 for a pint of Fosters, Strongbow or John Smiths on a Friday for students (or indeed any randomer that looks under 30), so we made the most of it.
After grabbing a bite to eat at home, we reconvened at the Parrs Wood tenpin bowling centre and bought a few pitchers of lager and two lanes. There were around eight of us there so we spent a good two hours bowling. Somehow I managed 3 strikes in the first game and 5 in the second, which is a rarity – maybe beer improves bowling prowess? This even included dabbling in different techniques, definitely not including using the finger holes – maybe we stumbled across a new method... We then faffed around on the pool tables until midnight and went into fallowfield for a couple of drinks – where we were able to admire some anti-Tory propaganda on the huge wall next to Sainsbury’s. I hit the hay pretty early for a Friday night though, at around 12 30. There was a good reason for this though – Warehouse Project on Saturday night, the best night in Manchester.
10am - 12pm ¦ Lie inI had a minor lie-in on Saturday and was only awoken before 11 by the sound of the door being hammered. Thankfully one of my other housemates stumbled out of his room to answer, which was fortunate because our brand new 36 inch HD-ready TV had arrived! I was feeling a little worse for wear from the night before though, so I lounged in bed and listened to 5-live sport and “Fighting Talk”.
12pm - 3pm ¦ Shop and relaxI went to the shop around the corner and bought eggs, bacon, bread, teabags and milk. I combined these to make an absolute feast of several bacon and egg sandwiches with a couple of brews on the side. At 1 I settled down in front of the new, insanely large television (relative to the size of our tiny living room) to watch Arsenal v Tottenham. I was delighted that Arsenal lost, as I can’t stand Arsene Wenger - mainly for his views on everything.
3pm - on ¦ Literature review / night outUnfortunately after the football I had to go into Uni, as Literature Reviews don’t write themselves. I also had about 150 papers to trawl through, so every spare minute is a useful one. I was also rather excited about Warehouse Project later so I used my good mood to bash out a good 7 pages. I managed to persuade myself to come into Uni today by leaving my laptop in my locker. Parking is also allowed after 12.30 on a Saturday outside the Smith building, so I could drive in and avoid the bitter breeze that is always steamrolling its way down the wind-tunnel that is Oxford Road.
From eight onwards the weekend got tremendously good fun. There were around 30 people I know going to Warehouse Project to see FourTet and Caribou – although I was going mainly for Theo Parrish. I wasn’t disappointed. We all met at around 10 at a friend’s flat in Fallowfield and downed plenty of beers. We then arrived at Warehouse Project at 11, where Theo Parrish had just started a 3 hour long set. I was over the moon, especially when he dropped the techno/jazz for 5 minutes and played one of my favourite dub tunes, the name of which escapes me. It was probably entitled “Kingston Curly Dub” or something along those lines. The night was tremendous and we all left pretty shattered at 5. I decided to go to a friend’s house for a couple more beers before hitting the sack at about 7 – I had to get some sleep so I would be able to get up and see the Rovers game the next day.