Month: June 2015

Another project that I was able to work on during my first week was PCR using DNA from different samples of ergot, a fungi that grows on plants such as blue grass and wheat. If ergot is ingested it can lead to vasoconstriction and can also cause hallucinations.

To perform the PCR, we had to combine primers, water, and master mix into each capsule along with the different DNAs. The master mix is a bright green color because it contains ingredients that help it sink into the gel later in the PCR. When pipetting all of the primers, master mix, and DNA together, you have to be very careful to not contaminate any of the capsules and to not mix the DNAs together. It can be tricky to remember what you put where with so many different tiny tubes!

After all of the solutions were combined, the capsules were centrifuged and then put into the PCR machine. This machine changes the temperature for specific amounts of time so that the primers and master mix can work to replicate certain sections of the DNA over and over. After the PCR machine was done, we created a gel using agarose. We put combs into the gel to make tiny wells. When the gel had firmed up, it was placed into the electrophoresis machine. Each DNA solution had to be carefully transferred into the wells of the gel using a pipette.The voltage of the electrophoresis machine was turned to 120 V. DNA has a negative charge so it travels towards the positively charged end of the machine. The agarose gel created a matrix for the DNA to move through which made the smaller pieces move farther than the larger pieces.

The sheet of agarose was carefully placed into a GelDoc-It Imager.The window of the imager allows you to see the gel under UV light.
The imager also allows you to record a computer image of the picture to examine for later use. 

This week I arrived in Madras to start my internship at the Branch Experimental Station. I was excited to start out the summer in beautiful Central Oregon!

One of the first projects that I got to work on after arriving was looking at how different treatments affected  seed-born carrot disease. First, we took carrot seeds that had the seed-born disease and separated them into giant tea bags. We had four different sections: orange, yellow, green, and teal. Each color section underwent the eight treatments: Non-treated, Hot water, Sporekill, PT81, FT33, KleenGrow, Oxidate, and Bleach.

We put all of the different trial solutions into mason jars and “brewed” the tea bags in the solutions to wet the seeds. It felt (and smelled) like we were making carrot tea!After the seeds had been ran through each of the solutions, they were carefully cut open and the seeds were laid out to dry underneath the hood. The hood pulls in air from the room, cleans it, and then pushes it over the work station. This made sure that the air going over the seeds was free of other bacteria that could contaminate them. 

After the seeds had dried, they were put back into envelopes and divided up into two sections. The larger portion was used for soaking the seeds in a phosphate buffer, which allows bacteria to live more successfully. The phosphorous-bacteria solution was placed into petri dishes containing a semi-selective media. Each trial from each color was put into 10 petri dishes. That’s a total of 320 petri dishes!

To plate the bacteria, a pipette was used to place the solution onto the dishes. Small glass beads were also put into the dishes. Shaking the dish back and forth moved the beads, which evenly deposited the bacteria over the media.The second reserved section of the seeds was used for germination. Filter papers were placed into the dish along with water. 25 seeds were placed into a 5×5 grid in each dish.

This was repeated 4 times for each trial of each color for a total of 128 grids.Now we wait and see how our bacteria grow and how the seeds germinate for each of the different trials!