Bumbling Along

By Sarah Wiseman

My third week began as the second had finished; with more RNA extractions (a task left over from the week before). I have to take samples from 24 different plants of different varieties, at different stages of maturity and have learnt the hard way that it’s a bad idea to tackle more than 4 at once, as the pipetting takes too long and the quality of RNA extracted starts to degrade.

PCRSarahMore excitingly, though with little success, I ran my first polymerase chain reaction (PCR) of the project. This is an important technique used throughout modern biology to amplify small quantities of DNA into quantities which are large enough to run tests with. However, you can’t just shove random fragments of DNA into the machine and hope that the right bits will be copied. Instead, we need primers which specify the regions to be amplified by the enzymes. Primers are short fragments of nucleotides, about 20 bases (the general term for the 4 different letters of DNA – A,T,G and C) long and bind to specific sections of DNA which complement their sequence. Primers must be well-designed, to amplify only the DNA you are interested in; so some work better than others. Whilst they can bind to DNA that they don’t perfectly match (and still allow the enzymes to make more DNA), the worse the match is, the less likely binding is to happen. Additionally, if the sequence is too general, non-targeted sections of the genome might be amplified leading to a confusing result.

PCR also often acts as a confirmation stage where we can check that things are working as expected – this is very useful when most of the time you are working with colourless and anonymous liquids! Before we designed our own primers, we trialed a set which targeted the rice version of the CKP gene to see if they matched up with the gene in wheat. These primers were already available to us and if successful, we wouldn’t need to go through the primer design process ourselves. Unfortunately, whilst some genetic material was amplified, sequencing showed us that the rice primers had amplified other random sections of the wheat genome instead of accurately copying the CKP gene as hoped. With no chance of other primers working, we spent a long morning working out which short sequences of bases would best amplify the CKP gene in wheat and have placed an order for their creation by Invitrogen – a specialist biotech company (other primer design companies are available…).

Since the primers were due to arrive in the next week, all PCR work was put on hold and I spent more time in the PGF. I felt rather cruel sorting through the Arabidopsis with my supervisor, weeding out the smallest plants for binning – those which were clearly not going to be ready for use in the experiments a couple of weeks later. We even composed a song from the perspective of the imperiled plants [to the tune of Bring Me Sunshine]:

“Give me sunlight, and some soil,

Lots of nutrients and water.

A little time, a lot of air.

Give me sunlight, give me soil, give me love!”

… you don’t have to be mad to work here but it helps!

On a more plant friendly note, I spent a happy afternoon helping my supervisor repot her wheat plants. They needed more root space and had to be prepared for their move into the glasshouse around the corner which can better accommodate the plants as they grow taller. Sadly, we only managed to re-pot about 200 out of 1000 plants, so we still have a long way to go….

Advertisements

Born to Pipette?

By Sarah Wiseman

‘So, what are you going to do after you graduate?’ A cruel question often posed by relatives, and sometimes even friends. It is particularly terrifying question for the soon-to-be finalist. Although a question potentially deflected by further study— ‘maybe a Masters or a PhD?’ is always going to be a better sounding answer than admitting that you don’t have a clue.

The Arabidopsis has just germinated, and is growing in one of the growth rooms beneath the Department.

The Arabidopsis has just germinated, and is growing in one of the growth rooms beneath the Department.

Though that does raise a second, slyer question ‘Am I cut out for it?’ Undertaking your own research is a completely different kettle of fish to attending lectures and a couple of practicals a week as an undergrad. With all that in mind I arranged (in an unusually proactive move) to spend roughly seven weeks working in Dr David Hanke’s lab, within the Plant Sciences Department at Cambridge. This blog within a blog will be a record of my time there, and proof that I didn’t just disappear in the Summer of 2014…

The Game is Afoot 

During this first week I have learnt many useful things; the location of the Growth Room (in the basement); how to sow hundreds of Arabidopsis seed (sterilise them in bleach then pipette evenly onto plates of agar); where the liquid nitrogen is kept (under lock and key behind the building) and more importantly, what I will be working towards over the next six weeks.

Harvesting and correctly labelling each variety – if you discover something amazing in a particular wheat sample it’s so good to anyone if you can’t remember which variety it belongs to!

Harvesting and correctly labelling each variety – if you discover something amazing in a particular wheat sample it’s so good to anyone if you can’t remember which variety it belongs to!

All of the experiments that I’m carrying out will be investigating the role of cytokinin binding protein (CKP) in dormancy. Cytokinins are a group of plant hormones involved in cell division/growth and CKP prevents their action – halting germination or growth for a given period of time. The CKP levels in different crops play a role in the security and reliability of our food supply. Low levels of CKP reduce the length of time which potatoes can be stored without sprouting. Whilst in wheat, low levels correlate with greater pre-harvest sprouting (when the grains of wheat germinate whilst still attached to the parent plant, reducing crop yield and making useless bread). My experiments will help provide evidence required to begin conventional breeding programmes in both wheat and potatoes, in order to produce crops with a higher level of CKP.

Grand unifying purpose aside, I did very little towards those ends, mainly collecting and preparing plant material – planting the Arabidopsis, ready for harvest in four weeks or so and picking ears of wheat. It was rather a lazy week, but you have to start somewhere!