Fungus Amongus

Some fungi may look the same but when looked at under the microscope they turn out to be totally different species because at the microscopic level their surface is extremely different. This is why I am sequencing part of five different funguses that I collected to verify their species. I have been working at the Forest Service Rocky Mountain Research Station, with my mentors Sara and Mee-Sook. They are leading me through this complicated project and teaching me tons of new things along the way. I have collected fungal conks which are the hard fungi that grow on the sides of conifer trees, and fruiting bodies, or mushroom heads. I collected five different funguses from Idlers Rest north of town.

The first step was to cut the fungi and get a “sterile” section that had not been exposed to the air. To do this we used alcohol and a heated ceramic tube to sterilize the tools between each cut of the fungi. We then put the section in a test tube with garnet pieces and little balls to smash up the cells to break the nucleus open to access the DNA. We put the test tubes in the “shake table” machine that shook the tubes all around, and then the test tubes were placed in the centrifuge. We pipetted off the top liquid which had the DNA. We then went through a series of steps to extract the DNA, which had lots of pipetting and centrifuging, and in the end we had straight DNA.

Polymerase Chain Reaction (PCR) is the next step. This is when a section of the DNA is duplicated many times so that it can be sequenced. We made a solution with the primers or the starting point of the sequence and the enzymes which bind the DNA, and the buffers stabilize everything. The solution was then placed in a machine that heats it up to 94°C for 30 seconds, then 55°C for 30 seconds, and 72°C for 30 second and repeats that temperature cycle many times. The 94°C will split the DNA in half, allowing the enzymes to start the process. The temperature is  lowered to 55°C so the primers will attach to the DNA template. After another 30 seconds, the temperature was raised up to 72°C for the bonding of the other base pairs. This series was repeated 5-20 times, depending on how much DNA was needed. After this, we took the test tubes with the copied DNA and prepared for electrophoresis.

Electrophoresis is the process of determining if any of the previous steps failed. To do this we used an agar gel, added dye with the DNA, and pipetted it into the agar gel with an electrical current. The DNA moves in the agar gel depending on how much DNA there is in each sample. The picture on the left shows a line across the top which indicates where the starting point of the DNA, and the other lines show how far the DNA traveled. We also used the TapeStation, which does the same thing except that it does it all electronically and a lot more quickly, as shown in the picture on the right.

From the electrophoresis, we learned that the process worked. Therefore, we will send off 4 of the 5 samples to be sequenced. This is what I have been doing in the lab so far this year. Check back to see what different species I may have discovered.

~Patrick