Team Phoenix


GTAC Day 1: Day 1 started off with a bang. We signed in at the front desk and met each other!!!

Group photo

The first thing we started off with was an introduction to each other, and then we went on an OH&S tour where we found out where all the fire extinguishers, safety exits, first aid kits and most importantly; where all the toilets were.

We then we went on to have a discussion/power point on what to include on our web blog, and how to format and structure it.

As we walked into the Doherty Lab, we had to make sure we had put a lab coat on and some gloves. We also had to make sure that nobody had brought in any food or water as per the lab requirements and rules.

Image of an optical microscope

Joseph and Danielle, two scientists from the BIO21 institute then talked about the life cycle of a fly and what species of larvae we were using and what mutation the larvae had for the second larvae we had the fly with the mutation Da6 KO, and about how they developed the wiggle assay, and how it is used in science.

We then got into our group and met our mentor Claudia, who talked us through the assay and made sure we were on the right path and were doing the right thing.

We were then taught how to use a micropipette, by attaching the pipette tip and correcting the level of fluid (micro litres) in the pipette. Then we were taught how to dispose of the tips by pressing the release button and putting them into the waste bin.

Once we had finished with that part of the assay we added a number of different insecticides to the larvae. We then went on to video 10 second increments of the larvae to see the effect it had on them over a period of time. We took a video at time point zero, 15 minutes, 30 minutes, 60 minutes and 90 minutes. The videos were then left over night to develop onto a heat map, to determine the Wiggle Index. Once all this data had been collected we put it into a spread sheet to generate a graph.

Day 2: 

We came in at 9:30 ready to start day 2. Josh and Danielle were ready and waiting in the Valente room. As we had put our data in to an excel spreadsheet on Monday and taken all the videos of each well for 10 seconds, all of our previous time points and data was ready to be developed. Overnight our videos were developing into 10 frames per 1 second, and then into heat maps once each of the frames had been established. With all of our videos developed into frames, and then heat maps ready to look at for analysing the wiggle index we started to normalise the data.

To normalise data, we used the formula RMR=   Raw Data At X Time point.

(RMR stands for Relative Movement Ratio)         Raw Data At 0 Minutes

 

Once we had a number, we rounded it to 2 or 3 decimal places to make it easier to read and plot on our graph.

Once we had normalised our data, we then had to graph it. The result are shown below in the graph.

 

 

2 Drosophila flies

Fly x40 magnification

 

 

 

 

 

 

 

 

After we had finished with our data from Day 1 we then went on to look at some mutant flies under an optical microscope. We were looking for physical features (Phenotypes) of the mutant flies that differed from the wild type flies. Some of the differences ranged from the length of their legs to the colours of the hair and colour of the eyes. After analysing the mutant flies and comparing them to the wild type flies; we talked about genotypes, phenotypes and what they were. We then talked about how we can predict what the recessive and dominant genes were using a Punnett square. We then completed an exercise based on the Punnett square and were able to predict what the offspring of the Wild type and Mutant flies were to look like theoretically.

After that we had a lunch break, and came back to an exciting session of using a Scanning Electron Microscope (SEM),  which use an electron beam to shoot electrons through the specimen, to give a 3D image, and incredible surface detail. We placed a carbon sticker onto the stage and then placed a wild type fly on to the carbon sticker, we then closed the door and evacuated all the out of the chamber. Except we had a problem it wasn’t able to. So we tried again but it didn’t work, so we talked and discussed about the other pictures that the other groups had been able to take using the SEM. Whilst we were talking about the pictures, time ran away from us and before we knew it was time to go home.

Day 3:

Scanning Electron Microscope (SEM)

After completing the DNA extraction, we were lucky enough that Frazer was able to get the SEM working again. This experience was amazing! We were able to see detailed surfaces of the wild type flies, these parts are varied such as the eye, back and wings. We noticed the damaged done by tweezers when placed onto carbon paper when ensuring they would stick. Frazer explained to us how expensive the machine was and how scientists use this machine to determine the different parts of a specimen.

 

 

 

As a result of the PCR machine not being turned on. This gave us some time to work on our blog for the GTAC work experience page on the website. This blog contained the information on what we had done for the day.

 

 

 

 

DNA extraction

Day 3 was just as action packed, if not more, as the previous 2 days. We started off in the Nossal Lab where we learned about DNA sequencing, hydrogen bonds and Nucleotides. We were then split off into groups to extract DNA from our mutant and Canton S (Wild type) flies from day 1 and 2. The first step of extraction was to add a Pre-Lysis Buffer (PLB) to a tube of Wild Type or Mutant Flies. This buffer strips away the fats and proteins of the flies. Proteinase K (PK) is next added to chop the proteins up, then we increased the rate of the reaction by crushing the flies with the pipette tip to increase surface area. The mixture is then incubated at 56 degrees for an hour to let both chemicals work. Lysis Buffer (LB) is then added to the flies to split their cells apart, this breaks the phospholipid cell membrane which assists in the extraction of DNA. They are then incubated again at 70 degrees. We then purified the DNA by putting it into a Centrifuge, a system that spins the mixture to remove wastes from the DNA sample using centrifugal force. This was repeated with different wash buffers to ensure we removed all wastes. After extracting the fly DNA, we next used Polymerase Chain Reaction (PCR), a technique that allows millions of copies to be made from one small sample of DNA by splitting it many times. To do this, we added primers (which bind to complementary nucleotides), Nucleotides (G, T, A, C), DNA polymerase. To speed up the process, by using enzymes of building DNA sequences and buffer (maintains the conditions for the enzymes to function) to our DNA samples. These tubes are then transferred to the PCR machine.

Day 4:

Today we began with completing the Gel electrophoresis. This was done by pouring a buffer onto the gel this is a salt water which is a good conductor of electricity. This helps the movement of the DNA, which continues the circuit allowing Gel Electrophoresis to take place. Once the buffer was poured above the gel, each member of the group pipetted 15 microliters into a well within the gel. This is a starting point for each DNA sample, when all samples were placed into their wells. We begin the electric current to begin the movement and replication of the DNA.

We allowed this process to run for 15 minutes.  After letting the gel electrophoresis run we placed the gel under a gel imager to be able to see the bands of DNA. Allowing us to determine whether our flies were mutant or wild type, we determined from the DNA samples that only one fly was able to be verified as a wild type.

PCR: Polymerase Chain Reaction

Gel electrophoresis

 

 

 

 

 

 

 

 

 

UV light gel imaging