I have finished my final PhD experiments and I am now full-time writing to finish my thesis. I have to submit by the end of January so my blog posts may be a bit less frequent over the next few weeks, but I will still be posting when I can. As a follow up to 5 useful, but tedious PhD tasks I thought I would describe some of my favourite experimental tasks (just in case I gave the impression that I haven’t enjoyed my PhD!) Some of the tasks are quite specific to my research area but others are much more transferrable.
Imaging calcium ions in root hairs using Cameleon
Calcium is a common signal in both plants and animals. One way of monitoring calcium ion levels within cells is to use Cameleon, a calcium-sensitive fluorescent protein (see my recent blog post for more information). I have developed Medicago truncatula plant lines that produce Cameleon protein, which means I can monitor calcium ions in cells of these plants in response to different treatments. I really enjoy these experiments because I get to use a fluorescence microscope to image cells and can “see” (through a computer calculating changes in fluorescence) the responses happening in real time.
Cross fertilising M. truncatula flowers
Each M. truncatula flower has both male and female sex cells (pollen and egg cells, respectively). Normally, like many plants M. truncatula is happy to self-fertilise with pollen from a flower fertilising the eggs from the same flower to produce seeds. Over successive generations self-fertilisation leads to offspring that are effectively genetically identical to the parent. This is handy for many studies because you don’t have to take genetic variation into account (like carrying out a study on identical twins instead of a pair of normal siblings). However, sometimes it can be useful for plants to cross fertilise, where pollen from one individual fertilises eggs of another plant.
Cross fertilising flowers involves several stages:
- Removal of pollen from the flower whose eggs you want to be fertilised.
- Depositing pollen from the other flower onto the stigma of the first flower (from here the pollen produces a tube to reach the egg cell).
- Careful labelling of flower so that you know you have cross fertilised it!
This is particularly challenging in M. truncatula plants because the flowers are small and so the procedure is carried out using a microscope and fine pointed forceps. However, I really enjoy the precise nature of the work and it is very rewarding to see a seed pod develop from a successful cross fertilisation.
When legumes form symbioses with nitrogen-fixing bacteria they produce organs called nodules. Some of my experiments involve treating M. truncatula plants with nitrogen-fixing bacteria and then examining the nodules that develop. I find the process of digging up the plants, washing them and counting the nodules rather fun!
Copying DNA by Polymerase Chain Reaction (PCR)
PCR is a way of copying “interesting” chunks of DNA to enable further studies. For an explanation of how it works click this link. Although I can find setting up PCRs tedious when many DNA samples are involved it makes my favourites list because I think it’s a really cool technique that makes a lot of other experiments possible.
The results from my experiments wouldn’t mean much if the data wasn’t analysed afterwards. For me this usually involves calculating averages or ratios, drawing graphs/tables and carrying out fairly basic statistical tests. I enjoy data analysis because the various actions can turn a bunch of numbers that are hard to interpret when you simply look at them into a format that highlights differences/similarities between sets of data.