This week, I have had the opportunity to do some work experience at the Institute of Zoology (IOZ) based at the Zoological Society of London (ZSL) London Zoo with my cousin who is doing a masters with the RVC and ZSL.
We arrived at London Zoo, Regent’s Park, through the hurricane, tackling London transport in chaos. As we entered the Wellcome building I discovered that behind the scenes at London Zoo there is a huge amount of research taking place. It is an active academic environment with lots of laboratories and facilities devoted to discovery. The IOZ supports a vast array of people in completing PhDs and this was what I was going to see. We climbed several flights of stairs, passing a doorway through which the experiments on live animals took place. I was strictly not allowed to go in here. The experimentation using living animals is regulated intensely, meaning that the majority of research does not use live animals. We then reached the herpetology department, where the all the amphibian and reptile research was based. My cousin hadn’t yet started her own dissertation however, spent much of her time helping her friend, Emma, to complete her PhD. I was introduced to Emma Wombwell and she soon started telling me about the research she was doing into chytridiomycosis. Chytridiomycosis, often abbreviated to Chytrid, is an infectious disease of amphibians which is currently a huge impact upon amphibians populations, especially frogs, across the world. Emma was looking particularly at Chytrid in the pet trade. She had set out to visit every pet shop in the country, swabbing all their amphibians then bringing back the samples to analyse for traces of the chytrid fungus, Batrachochytrium dendrobatidis. She hoped to be able to track where the amphibians had been imported from, and hence shed light on the areas of the world in which chytrid was most severe so that further research could be focused here.
Today she was going to be extracting DNA from samples she had already collected, ready to be used for real-time PCR which would test for chytrid. She showed me how to use all the equipment in the laboratory and gave me a chance to try a few things out myself. The steps were carried out as follows:
- Organise 48 samples to be analysed.
- with the huge number of samples she has collected, it is important that she carefully keeps track of which sample came from where. Every pet shop has a number, and every sample taken there also has a number. Here we were extracting DNA from swaps from pet shops 678, 84, 676, 453, 666 and 7002.
- it is important to use 48 samples because most trays are arranged in rows of 12 and the centrifuge has room for 24 tubes.
- Place 0.03 – 0.04g of Zirconium/silica Microbeads into centrifuge tubes.
- Pipette 60µl Prepman ultra into tubes.
- PrepMan® Ultra Sample Preparation Reagent has been developed to prepare DNA ready for PCR as it inactivates PCR inhibitors such as lipids.
- In a fume cupboard, cut off the tip of each swab, using a scalpel on a petri dish and place one swab in one, correctly numbered, tube.
- it is advised to be done in a fume cupboard to avoid the spread of the fungus from the samples into the lab.
- to avoid contamination it is important that a new scalpel blade is used for every swab, however the same petri dish can be used up to 4 times, as long as it is turned round between swabs so a different area is used.
- Homogenise the samples using a bead beater
- Centrifuge the samples for 30 seconds.
- Homogenise again.
- Centrifuge again.
- Heat at 100°C for 10 minutes in a heat block.
- this causes the DNA strands to separate by breaking the hydrogen bonds between base pairings.
- Cool for at least 2 minutes
- Centrifuge for 3 minutes
- this causes the heaviest organelles and other parts of the sample to be pushed to the bottom of the tube, leaving a supernatant containing the DNA.
- Pipette as much supernatant as possible out of the tubes, ensuring that the cotton from the swab and microbeads remain in the tube.
- Freeze samples until PCR.
I had a fantastic day today and really enjoyed learning about the processes involved in extracting DNA. It was brilliant to be able to apply the knowledge I have learnt at school about the structure of DNA to help me understand the steps which we were going through. I am looking forward to seeing the process completed using PCR, and uncovering some results.