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.
More 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….