Tag Archives: Everyday lab

Adventures in protein land part 2: Cytosolic protein extraction

So this is going to be more like a lab book entry than anything else, but I’m finally starting to get my head around some of this protein malarkey and figured I should write it down in multiple places!  Continue reading

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Adventures in protein land

I’m not a protein biologist.

I wouldn’t usually describe myself as a geneticist either, unless trying to clarify what it is that I spend my time doing, in the same way that for years I would describe myself as playing the violin rather than as a violinist, because that seemed to indicate some level of skill that I didn’t feel I yet had.

But I’m definitely not a protein biologist, because I did no protein work as an undergrad, and I didn’t sign up to do a protein based PhD, and there’s only one post doc in the lab who has the first clue about proteins and – while she is possibly the most helpful post doc ever to cross my academic path – there’s only so much help someone can give you when you don’t know what questions to ask.

Yet in spite of this, I appear to be in a situation where doing protein work is inevitable. A few months ago I learned about PAGE gels and Coomassie blue staining and Western blots. Now, I must actually start extracting protein from real live plants, as opposed to my nice-n-easy-burst-like-a-bubble-E. coli. It turns out that this is rather more complicated than I had hoped.  Continue reading

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Starting Grad School: The beauty of a clean lab book

Occasionally, I have the realisation that I am not entirely sane. Not in a catastrophic hearing-voices type way, but when I talk to myself and then remember that this is not normal. This morning I arrived at the sports centre to discover that circuits wasn’t on, and I would just have to go run in the gym instead. Upon finding my mp3 player in the bottom of my bag, and realising that I had in fact remembered to put it on hold (therefore meaning there was enough battery to get me through a 40 minute hill slog) I smiled and mentally called myself a good girl . Continue reading

The Ramen Diaries, Part 4: Oh god, it worked?!

This afternoon as I walked back over to the post-PCR lab (after more ramen – today’s was pork) I thought sadly to myself about how far I have come. When I started Grad school, full of enthusiasm and confidence, if something like a PCR reaction didn’t work I would instantly start looking for what might be wrong: what could I do differently next time. The idea that ‘it just didn’t work’ was completely alien to me. Now I know better. Primers that look perfect could easily correspond to a repeated sequence. Reaction conditions that are completely contrary to everything I know about PCR design can produce much cleaner results than a perfectly designed reaction. I’ve learned to loathe PCR, and along with it, expect that any ‘completely new’ reaction just won’t work.

Continue reading

The Ramen Diaries, Part 3: A cunning plan

On today’s postgrad menu, Tom Yum ramen. Why are all of my noodles so spicy?!

Supervisor has suggested a cunning plan to solve my qPCR woes: put DNA into a regular PCR for 5 cycles first, then transfer to qPCR afterwards. While this doesn’t really solve the problem that after X number of PCR cycles we expect an artefact to appear anyway, it does have the advantage that the machine will actually call the Ct value. (Anything over 35 cycles, even if it’s ‘real’ isn’t called by my software, and is just 35+).

So far, so unsuccessful: but I tried using the qPCR primers with a high fidelity Taq that I haven’t tried them out with, so it’s entirely possible that the reaction just wouldn’t work like that. Tomorrow I’ll repeat it with my regular everyday Taq.

Annoyingly, somebody from another department keeps calling up wanting to use our qPCR machine, since theirs is bust, and then never turning up. I have to plan my labwork around somebody else, but then lose big chunks of every day when the machine is “full” and by the time I know it’s not it’s usually too late! GRRR.

The Ramen Diaries, Part 1: qPCR failure

Dear Diary,

Today I became a proper postgraduate student (after 3 years two months and about five days) by purchasing Ramen noodles for the first time ala PhD comics:

https://i1.wp.com/www.phdcomics.com/comics/archive/phd070811s.gif

Unfortunately lab work is less successful. There are about six weeks left until PAG and I desperately need to crack on and get this data. Having finally – I hoped – solved my qPCR non-specificity woes by spending inordinate amounts of money on LNA oligos, which – at 65C – became specific between homoeologues, I am rapidly finding that they are so specific in a qPCR reaction, even at 60C, that they will not make it to threshold fluorescence without using more cDNA than I can possibly afford to put in a reaction.

This is what I believe is called a Catch 22.

I am now drowning my sorrows in salty, MSG-y goodness and dreaming of a life of running routine experiments instead.

love,

baking biologist

When science gives you lemons… find out if lemonade exists

Some days, life throws you curve balls. The lights are all red; Sainsbury sell out of orange milk; the weather forecast is clear and then it heaves down. (Seriously though, this is Britain. Who am I kidding? If I leave the house without a raincoat, it’s my own stupid fault).

More frequently – in my case at least – science throws me curve balls. In fact, what am I saying? I can’t even remember the last time science threw me a straight ball.

For the last two days I have been sat sadly staring at this data. There’s a special kind of paralysis that comes upon a PhD student when an experiment might be failing and they’re just not sure: it’s the ‘I won’t know until I’ve finished it… but I don’t want to waste my precious precious samples if I could fix it instead’ sort of feeling. This isn’t really a data set yet, just two qPCR plates. The first one looked a bit funny, but the standard curve just plain didn’t run, so I was planning to discard the plate and run a new replicate. Then the second plate came out looking exactly the same. (The green line is the same samples against a different housekeeping gene).

I am looking at the expression of a gene in the wild type plant, three single deletion lines and a double deletion. (I work with a polyploid, so by crossing and then selfing the deletion lines, multiple deletions can be stacked). I expected that either the plants would show reduced expression (because they are missing a copy of the gene), or that they would be the same as the wild type: because the plant was compensating for the loss.

These are the differences in Ct values between the gene of interest and the housekeeping genes: i.e. how many more cycles did it take for the sample to fluoresce. A value of 3 is more or less equivalent to a ten times difference in expression, so really I guess I was expecting to see a difference between lines of 1 cycle (or about a third). High values mean low expression. Low values mean high expression.

Deletion line 3 is doing what I expect it to: the delta Ct value is higher, because expression of my gene is lower. So far, so good. Expression of Deletion line 2 is static. Okay, so this one is compensating. Also good. The two deletion lines are different: that’s interesting.

Uh… hold on a second. What is deletion line ONE doing? And more to the point, what is the DOUBLE deletion line doing?

*cue freak out*

How on earth can a plant that is missing a copy of a gene then be producing MORE of the transcript associated with that gene?!

For the last day I have sat and pondered. And by pondered I mean I have tried to write an application, I have marked some A-level work, and I have tried to write a chapter for FastBleep Biology that I may never finish at this rate. Today, I cracked. I told my labmates – in a slightly hysterical voice – what my data looked like.

And without batting an eyelid, one of them said “You’ve deleted a regulator.” … I’ve pardon me? “You’ve deleted a regulatory element. Gene expression is no longer being suppressed. In fact if the double deletion is doing the same thing then that sounds even more like the answer.

There’s an answer. Not only is my science not failing dramatically, but there may even be something interesting going on.

Hello science. We meet again.