Category Archives: In the 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

The Ramen Diaries: Part 8 – Protein Extractions

After a week off with flu, there’s just no avoiding it. Today I have to get back in the lab and do my first ever protein extractions from a plant. I have a protocol, that I am assured works, and no reason to suspect it doesn’t. And I have been sat here for 54 minutes doing admin instead of even going to the greenhouse to get my plants. Looks like the stage fright is back. Le sigh….

Tales from the Teaching Lab: Early misconceptions in genetics

When I started this blog, it was supposed to be a news blog: me getting to grips with and explaining new papers that I found interesting. Recently I’ve been thoroughly uninspired, but trying not to resort to writing more opinion pieces about how to survive Grad school (even if that does seem to be what I do best).

Nevertheless, it’s been over 2 weeks since my last post, and while demonstrating yesterday, a familiar itch beginning to niggle. Whereas in previous years I’ve done a more-than-normal amount of tutoring and demonstrating, this year I’ve only covering a small handful of lab classes, one of which is Introductory Genetics. The class is pretty simple. Students are given a variety of mutant Arabidopsis that they have to phenotype, in addition to the F1 and F2 of back crosses to the wild type and crosses between mutants. They are trying to find out whether the mutations are single gene traits; dominants or recessive; and whether multiple mutants are caused by mutations in the same or different lines.

This is the third year I have demonstrated for this class, and the previous year I marked the open book exam that students take at the end, without having demonstrated for the practical, so I’m probably more familiar with the class than anybody except for the professor teaching it. Along the way I have had to do a fair amount of mental gymnastics, but I’ve also picked up on a few of the most common misconceptions that students hold. I actually clearly remember as a first year undergraduate being confused by the term “wild type” because nobody had explained to me that this was a technological term for the working copy of a gene: I thought it meant the type of gene that animals had in the wild. Which is fine, until you start thinking about mouse or Drosophila genetics!  Continue reading

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.  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 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).  Continue reading