The Ramen Diaries Part 7: Or Twitter, a cautionary tale

So if you’ve read the last few posts then you will know by now that I spent last week at a big genomics conference in San Diego, called PAG. You’ll also know that, while I was there, I encountered some people who walked out of talks, yawned and slept their way through talks, and just plain talked through them.

The Ramen Diaries, Part 5: Where I throw a tantrum

When I was in my teens, the thing that I loved more about science was the opportunity to learn something new every day. I loved that what I thought I knew was never exactly true, and hated it at the same time. I wanted to know what we’d be told in our GCSE classes, or our A-level classes, or our undergraduate lectures long before I was old enough for it to be on the syllabus. I had this idea that if I kept studying science for long enough, eventually it would make sense.

It will never make sense. 

The Ramen Diaries, Part 2: Think Fast

So qPCR is still kicking my ass. Integrated DNA Technology actually tweeted me back in response to my moaning about LNA oligos, which made me smile even if it wasn’t especially helpful.

Essentially: my primers may be awesome (provided I can acquire an unrealistic amount of DNA) or they may be unusable. I can’t tell without testing them with a stupid amount of cDNA. (Which I don’t understand, because they worked in regular PCR just fine). 

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:

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

Idiot’s guide to Western Blots: Part 1

Having not touched them since … June maybe? Perhaps even May … I’m back to doing Western Blots, a technique that I wasn’t amazingly confident with to begin with. I did a dry run with samples that didn’t matter on Friday, knowing that Iw as likely to make tonnes of little mistakes all over the show, forgetting things that once seemed too intuitive to write down. What I need, I thought to myself, is an Idiot’s Guide.

There’s something that bothers me about Idiots’ Guides: They never tell you where something will go wrong. Or what will happen if it does. So here I present the Baking Biologist’s guide to Western blotting: The things that can go wrong, why they will go wrong, and whether to carry on or just scrap the whole thing.

Starting Grad School: Stress management

Given that it’s that time of year when both my new undergrads and the new crop of postgrads appear like magic in the department, I thought I might scribble some thoughts about life at the start of Grad School. Call it egotistical, but given some of the rubbish I’ve been through in the last 3 years I feel like I might have a thing or two to share that newbie postgrads could find useful.

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.

A note from the lab bench

Dear internet,

It is infuriating to discover you have slipped up somewhere and ended up with next to no DNA or RNA or whatever at the end of your prep.

It is even more frustrating when you had planned really well and done everything to the letter.

But the most frustrating thing of all must be to be certain you have done it all right, have it all go wrong anyway and then discover someone else in the lab already knows why. 

I have (had? *sadface*) some RNA. I DNA-ase treated it prior to using it in qPCR. It needed cleaning up.

In the previous incarnation of this experiment I cleaned up the RNA (Qiagen RNA MinElute) then DNA-ase treated it. (Turbo DNAfree, in case you’re interested).

Except that the more I thought about it the more I worried that I was leaving residual DNase behind. So this time around I used the Turbo DNA-ase and then cleaned it up using an RNease Mini Kit. Exactly like it says you should. And all of my beautiful RNA is… well not gone. But coming out at ~50ng / uL. FIFTY. What the actual hell?! The other way around I got nearer TWO THOUSAND!!

Now obviously a MinElute elutes into 14 uL not 30, so it’s only natural to expect the prep to be half as concentrated: let’s say it was a pretty rough prep that would have had 800, then I might expect 400. But fifty?! Absolutely gutted and so unbelievably cross with myself for throwing all 14 samples at it at once.

But the most annoying part of all? Another PhD student in the lab figured this out about 2 months ago. Turbo DNA free and Qiagen RNeasy mini just aren’t compatible with one another and as far as I can see nobody has ever made a note of this.

Yours grumpily,

Baking Biologist

#summergoals: End of July evaluation

A few weeks ago I decided to get a handle on my summer by setting myself some goals (thanks to  Flora Poste for giving me the idea!) . I’m a very target driven person and also prone to floundering without a schedule (someone remind me why on earth I wanted to do a PhD?) The aim of the game was to make SMART goals (Specific, Measurable, Attainable, Relevant and Time-bound). In other words I set myself a whole bunch of tasks to be achieved; some big, some small; that would help my PhD in a noticeable way, and that were supposed to be complete by certain deadlines. The end of July was one of these deadlines.

My first recap of #summergoals was pretty successful. The end of July… less so.

#summergoals: End of Week 1

At the end of last week I wrote a post about my #summergoals: aka the baking biologist’s attempt to be organised, and not have her life taken over by generic moping, procrastination, and political machinations. (Did I mention I’m an evil genius?)

As week 1 draws to a close, it seems sensible to establish how I’m doing so far (aka give myself a kick up the behind).