Tuesday, 28 July 2015

All hail the God of PCR

At a particularly low moment in my PCR optimisation process, I created a shrine on top of a cupboard. It was dedicated to a god of PCR. I'm against the non-critical thinking that leads to religion and faith, so you can probably imagine how low I had to be to create a shrine. But I thought that if, despite all evidence and logic, there was a god of PCR...I had better start worshipping it.

Since then, everything has pretty much gone to plan. I have had more samples work than not work, and I've begun to assemble a pretty comprehensive tree of Cantuaria. It has mostly shown me that the current taxonomy of Cantuaria places them into different taxonomic groups (species, genus) than their genetics do. But there is still a bit of work to do with BEAST, the program I am using the build the trees.

I've decided that optimising the stubborn samples that I have left (which all seem to require completely different PCR conditions) would be a waste of time and money. I have enough for my tree, and it would be nice to get them all to work, but I was meant to have completed this six months ago. I've got three new genes to work with as well (thanks to a friend) which I'm going to use to create a different phylogeny with fewer samples, but more sequences per sample (an idea which I picked up from the Evolution conference). That is not going to involve optimisation, though. If stuff doesn't work, screw it, I'm out of there. I have more ecological fish to fry.

So, all that I have left to do in the lab is...
-extract DNA from the 43 remaining spiders that I have collected
-PCR for all three genes these 43 spiders
-CO1 PCR a few more samples
-PCR the new 3 genes (I'm aiming for 14 sequences - two from each major clade).

If the PCRs for the 43 remaining spiders don't work, I'll try adding BSA (a magic liquid extracted by squeezing cows), doing a gradient PCR, and using MyTaq, which was recommended to me by another person who studies the same family of spiders (but seems a little sensitive for most of my samples). But that is the limit to which I will go to make them work.

There is a light at the end of the tunnel. Wish me luck!

Saturday, 4 July 2015

Evolution Conference, Brazil 2015

The perching birds (passerines) decided to have a conference to display their various adaptations. Nightingale went along to see what the other birds had been up to. However, when he got to the conference, he could not see any other nightingales; the only member of his family was a blue flycatcher, with his striking cobalt and rust plumage. In fact, none of the other passerines at the conference looked much like a nightingale at all. They all seemed to be brightly coloured.

Nightingale went to some of the displays the birds were giving. Most of them comprised dancing, or showing off their bright plumage; manakins were moonwalking and jumping up and down, birds of paradise made their usual elaborate displays, and other colourful species simply talked about how their attractive feathers helped them to find quality mates. Nightingale soon grew tired of their splendour, and wished his plumage was comparable.

At one of the mixers, Nightingale met a lyrebird. He was just as brown as Nightingale, but with long, elegant tailfeathers. "He's not so gaudy as the other lot," Nightingale said to himself. "He looks better than me, but he's still not quite there yet!" It made him feel a little better to see a brown bird. He wandered over to the lyrebird and bowed.
"Hello there," said the lyrebird. "I am Menura. What genus are you?"
"I'm Luscinia," said Nightingale. "I specialise in song," he added.
"Oh, a songster!" said Lyrebird, obviously elated. "Fantastic! There's only you, me, and Songthrush over there."
Nightingale went to Songthrush's display, and was comforted: the thrush's song was not nearly as beautiful as his own. But when he went to see Lyrebird's session, Nightingale's heart sank: the lyrebird began with a lilting melody, then incorporated beautiful and exotic sounds from the human world, and finished by imitating every bird that had come to watch him.

Nightingale was so flustered that he wanted to leave the conference. He felt so inadequate that he didn't look where he was going, and bumped into a sparrow on the way out. 
"What's wrong?" asked the sparrow, her face full of concern.
"Oh, I'm supposed to be a passerine, but I can't possibly compare to the others with their bright feathers and wonderful dances - even the dull ones can sing better than I can!" Nightingale sobbed.
"Ha! Those posers," said the sparrows. "Don't believe for a minute that you need bright colours and gaudy displays to succeed as a passerine. Sure, they work for those who use them, but you won't catch us growing sparkly feathers - we'd just get eaten!"
More and more sparrows began to crowd around the poor, crying Nightingale. They offered him tissues, and one put his wing around Nightingale's shoulders. "There are so many of you," Nightingale gasped.
"Yes! We're one of the world's most successful families of bird. We have colonised every continent except for Antarctica, although we've had a bit of help from the humans," said a sparrow.
"But...you're so dull coloured!" Nightingale said.
"Success isn't much to do with how bright your plumage is," said another sparrow. "It's more to do with your biology as a whole. If you've got a good well-rounded ability to adapt and survive, you'll do well, young nightingale, and have a brilliant future!"
One by one, the sparrows went back into the conference, focusing on displays of behaviour and foraging rather than the showy displays that Nightingale had been going to. Nightingale followed the sparrows, learning about their natural history, and took home plenty of ideas about how his lineage might adapt to the challenges of the future.

Bird phylogeny by Jetz et al. (2012, Nature). Species within the black line are passerines.