Category Archives: In the lab

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.

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Molecular Biology is like baking because…

If you are a sufficiently regular reader that you have ever googled me directly to get here, you will know that I am not the only baking biologist on the internet. In my haste and excitement to found my blog I neglected to realise that there is another baking biologist over on blogspot. Luckily, she mainly bakes, and I mainly biologise, so there isn’t too much cross over. Then there’s Sugar Scientist  and Domestic Diva MD and of course Dr Isis, Domestic Goddess.  It seems that baking is quite the past time for scientists in general and biologists in particular.

We have a running joke in the lab that there should be a list of questions that we ask to interview candidates before accepting them as members. It’s really a long list of things my PI dislikes about us (in a Grumpy Old Man way, rather than a genuinely-frustrated by way). The current list stipulates that new members of the lab:

– Must not have coloured hair (in the past 12 months alone mine has been purple, scarlet, royal blue and turquoise).
– Must not own cats
– Must not knit
– Must not have or use a mobile phone, especially in the lab
– Must be strongly opposed to the construction of pylons
– Must not read fiction
– Must attend the lab day out (my PI’s favourite day of the year is when none of us come in and he can have the lab completely to himself)

And the most recent addition to the list (thankfully, added after numerous successes not failures) is that new members of the lab must demonstrate their accomplishments as a baker. Because, ultimately, molecular biology and baking are very similar to one another in pretty much every respect other than scale.

Don’t believe me? Well let me explain…

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. Continue reading

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

The mysteries of lab packaging

I’m sure I’m not alone in having regularly received lab supplies – especially enzymes – shipped in bizarrely large boxes. Today’s offering, however, really takes the biscuit:

This box (with autoclave tape for scale) contained four of these tubes (with my hand to scale). In fairness, it was shipped over the weekend, but all the same that is a shedload of dried ice for four very small tubes!

Amusingly, today I also received this.

This is also a qPCR enzyme and also says that it should be stored at -20C. This one however was shipped in a plastic bag with a (completely melted) ice block. Fingers crossed it still works… not planning on using any precious samples to test it though!

Lab fright, and how to fight it

If you have happened to glance at the About page, you’ll know that when the baking biologist isn’t doing biology or baking, she also sings. What you might not know is that when she sings she gets major stage fright. Those who are not involved in any kind of performing arts might not have ever experienced stage fright. It’s not about being scared. It’s about the racing pulse, the sweating palms, the ringing in your ears as the panic rises…

But on a stage is not the only place the baking biologist gets stage fright: she gets it in the lab too.

When I walk into the lab there is sometimes a little voice that tells me You don’t belong here. It’s one part a Professor with a very dry sense of humour, who was single handedly responsible for me dropping molecular biology after first year (only to decide when I graduated that I wanted to do a genetics PhD….) It is one part a real badgerwaffle of a scientist that I had to work with after graduating, who went as far as telling me to do things wrongly just so he could enjoy watching me screw up. But though there are specific reasons that lab work unnerves me I don’t think I’m alone in finding a molecular biology lab a scary place to be, and I don’t think I’m the only one whose imposter syndrome stretches to the bench.  Continue reading