Wednesday, July 31, 2013

Leeah: Week 4

          Primarily the focus for this week was on molecular work. Over the past month I have frozen a plethora of pilidium larvae. On Monday I ran the biggest extraction I have ever done before. This DNA extraction consisted of sixteen previously frozen individuals. Terra had some business to attend to on Tuesday and I was left to my own devices for most of this week. She gave me a list of high tides and low tides so I could plan some plankton tows. I was also entrusted to run a PCR on a few samples she had left with me. It was exciting to see the pictures she had taken of the larvae and have a face to match with the glowing band on the gel tests.
           I know that it is rather confusing to explain molecular work here. I now have access to pictures which will help explain the process.
As I mentioned before, we use a product called Instagene for larval samples which has little beads that hold everything but DNA. The beads need to be stirred using the Nuova stir plate as shown below.

A pipetman is used to transfer the Instagene to the larval sample.

This is one of the incubators used in the DNA extraction process

After incubation, the samples need to be centrifuged so the beads will accumulate at the bottom of the tube.

Then the supernatant liquid which contains the DNA can be removed and put in a new container.

Extraction complete now moving onto the PCR.
I split up the samples based on the gene regions we study, 16S and CO1. I had 16 samples and now I have 32!

I put a few microliters of my extracted DNA within and some Master Mix to get the reaction going. Then it goes in a machine which is has programs that run at temperatures specific to the gene regions and the reactions.

Time for an agarose gel test. This process is called gel electrophoresis.
This is the gel rig that holds the agarose containing a chemical that binds to DNA called Ethidium Bromide.

The gel is melted and then poured into the rig. It is then allowed some time to set.

Combs help to mold the gel and provide a place to load the samples. It is important to check for any bubbles between the teeth of the comb.

Then buffer is added to the rig. It must have complete coverage over the gel.

Then the combs are removed so we can load the gel with our samples.

Small quantities of the samples are added to each place left behind by the comb. A ladder or molecular weight size marker is also inserted as to show the quantity of DNA when the gel is placed in the imager.

The rig is then hooked up to a current, running from black to red, which will pull the negatively charged DNA to the positive end.

After about 30 minutes, the gel is ready to go into the gel imager.

Agarose gel test completed! Now the troubleshooting begins.

 On another note I have taken a break from drawing worms this week. I was excited to find a Sea Otter skull in the Birds and Mammals classroom and I decided to draw from it. One interesting thing about otters is that their lower jaws cannot detach from the upper part of the skull. Evolutionarily this makes sense as otters routinely eat hard kinds of prey which would be easy to dislocate one’s jaw on. The upper image of this drawing depicts the underside of the otter’s skull where the mandibular condyle on the otter’s jaw is completely interlocked with the mandibular fossa. The scientific name for Sea Otter is Enhydra Lutris which literally translates to “Otter in water”.

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