Day 16 – The Meat
We anxiously awoke once again from our Mweka dormitory rooms prepared for the busy and important day ahead. Today was crucial, not quite the day of reckoning but almost, because this afternoon we were performing the DNA extraction protocol.
After first awaking from our slumber we ate a quick Tanzania breakfast of bread, hardboiled egg, and a pancake like crepe. Following the small meal we moved to the laboratory to begin the activities for the day. The first activity was to do an overview of yesterday as well as an overview of the extraction protocol. It took us about an hour to perform this overview but the time was well worth it as it prepared all of the participants for the rest of the day. It was Dr. Vavra during this period that was performing the teaching and so everyone else prepared the meat samples for the participants. This was quite interesting as we prepared three different samples: two fresh and one jerky. And prior the actual placing of the samples into microtubes we asked the participants to “guess” what type of meat it was. This was quite an interesting test as each of the participants are experts in the field and have seen a multitude of meat samples. Thus it was quite interesting when there was such a wide variety of guesses. Throughout the guessing we asked how they were creating their hypotheses. There were several main factors that the participants used to determine the species:
- Fiber size to narrow down the size of the animal
- Color of the meat to narrow down the species
- Smell to tell the difference wild and domestic
- Quantity of fat on the meat to determine wild or domestic
Sorry to those reading but we can’t post what the actual species we had are but we did have a domestic sample, a bushmeat sample, and a jerky sample. Please check back on a later day, after we have revealed the species to the participants, and the information will be online.
After the species hypothesizing the participants together then began the DNA extraction. In this step the participants added placed their sample in both a dry tube and a tube filled with isoproponal, to represent an in the field sample. The samples were then homogenized and a buffer was lysis buffer was added to break apart the cells and proteinase K to remove excess protein. The samples were then placed into a heat block to digest for 2-3 hours. (Note: This procedure is over simplified and is not word for word accurate. Please email if you have more specific questions on the protocol.)
At this point all of the groups had their samples in the heat block for digestion and we took a short tea break. Now, just before lunch, everyone was regrouped back into the lab to for a short explanation on the rest of the DNA extraction. In this quick explanation Zac described the MagnaRack, part of the Charge Switch kit, and how it would work in the DNA extraction process. Zac also described the pros and cons of the system, such as the fact that it small and portable compared to the full sized centrifuge but it was a bit more time consuming. Nonetheless, Life Technologies has done an amazing job simplifying the equipment so that it can be used anywhere, such as a Spartan laboratory in Tanzania. By the end of the explanation the participants were ready to move on but a quick lunch was in need.
Lunch ended and the participants were then split up based on the their samples, 1, 2 or 3. These groups then alternated between either the next steps in DNA extraction or a discussion on PCR, which normally had two groups.
For the DNA extraction we began to use the central component of the Charge Switch method, the MagnaRack. So for the participants moving on to the next step they would first began by adding a lysis buffer to break apart the cells. Once the cells are open you first must remove the RNA using an RNAse buffer. After completion of these first two steps you then add positively charged microscopic magnetic metallic beads. As we know DNA is negatively charged and so the DNA and beads bind together. With the DNA bound to the beads you can then place a magnet next to the container and all of the beads will clump next to the magnet. So, the DNA is attached to the beads and the beads are contained in one small part of the container. At this stage it is just a matter of adding a series of wash buffers and continually removing the liquid, to remove other organelles, to extract the DNA. The final step though is to separate beads from DNA. This is done through Elution Buffer (EB) which removes the charge from the beads. With the DNA and beads separate the magnet is added to pull the beads to one side. The EB will then contain your extracted DNA. Sorry if it is a bit complicated to our readers but the entire procedure is a bit long. If you are interested in learning more please email Zac.
While some participants were working on the DNA extraction the rest were learning about Polymerase Chain Reaction (PCR). PCR is an incredibly important step in DNA sequencing. The basic idea of PCR is you place your DNA product into a thermocycler with Thermus aquaticus (Taq) (An enzyme which binds the primers), primers (to start the new DNA strands), and nucleotides (the A’s, T’s, G’s, C’s that build the DNA strands). So, you place all your materials into the thermocycler and then by adjusting the temperature you copy your DNA. Thus, the process is like a DNA copier, going through 35 cycles. By the end of the process you have billions of copies of DNA. This is important because if you do not have enough DNA you can not sequence.
This was about all we did today, but tomorrow we will actually be performing PCR. Exciting.