Tag Archives: qPCR

Rusty Research: Fighting Bread’s Biggest Bad-guy

This has been a bad year for farmers: last year’s wet summer and then the cold winter that just won’t end have scuppered one harvest and probably knocked this year’s right down too. Even when conditions are more ideal than they have been this year, farmers and breeders fight an uphill battle trying to prevent a significant proportion of the crop being lost to various pathogens. When it comes to wheat that means rustblack rust, brown rust and yellow rust. Where it strikes, yield losses are likely to be around 20% in susceptible varieties, and the problem is getting much worse. Most resistance to black rust (Puccinia triticina) is caused by a single gene, which a new resistant kind of rust (Ug99) managed to overcome in much the same way as MRSA became resistant to methicillin in our hospitals.

Close-up of wheat leaf rust (”Puccinia triticinia”) on wheat. Photo by James Kolmer. http://www.ars.usda.gov/is/graphics/photos/jun06/d519-1.htm Image Number D519-1 PD-USGov-USDA-ARS

Now scientists from Norwich, Cambridge and the USA are trying to find out how some kinds of a similar disease, yellow rust, (Puccinia striiformis or PST) are able to overcome the plant’s natural defences and infect.  Continue reading

The Ramen Diaries, Part 6: Where I work on a Sunday

When I started Grad School, two of my best undergraduate friends and plenty of acquaintances were already at the end of their first years of a PhD. I think just about everyone from my circle of friends who went directly to PhD without going via a Masters stayed at our alma mater (which, I suppose makes sense: somebody who already knows you and your work is more likely to take a chance on you, and somebody who has already found a lab they want to work in is more likely to take time out of studying for finals to apply). 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

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