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It Happens

Tuesday, August 1, 2017
It Happens
Five snapshots during the process of DNA, start from the top-left and go clock-wise until you hit the bottom-left.

Hi everyone!

 

I previously published a blog post called Going through the Alphabet, where I talked about the trials and tribulations that I faced during the process of nailing down my project.

 

Since then, I’ve learned that the same persistence is required for the data collection stage.

 

My project has three major components: 1) testing Mongolian Oaks for bacterial leaf scorch by extracting DNA and using a polymerase chain reaction (PCR), 2) determining moisture content by measuring fresh and dry weight, and 3) determining the percentage of foliar Nitrogen present.

 

The first and third components in particular require several steps and depend on a variety of factors. Extracting DNA and using PCR involves a tremendous amount of pipetting small volumes of liquids. To visualize your results, you must run a gel electrophoresis, which can be finicky at times. In addition to the hours of benchwork required by these three steps, you must account for the time required for instruments to run. Once you have amplified your DNA using PCR, you must put your samples in the thermocycler for about two hours. Gels take at least an hour to set up and run. Use of instruments is also limited by availability and space within the instrument. I have fifty samples, which translates to three rounds of DNA extraction, four rounds of PCR, and three gels. The number of steps within each of these three stages, coupled with the repetitions required by the number of samples I am testing and the mistakes that I have made with pipetting, results in a very exhausting and time-consuming process.

 

 

 

 

A beadbeater whirring as the tubes inside are shaken until they are just a blur.
The new beadbeater in the herbarium lab that I began to use halfway through my extractions, as the old beadbeater did not work very well.
Alyssa Gao

A machine, its screen lit up, with a piece of paper draped over the top.
The thermocycler that the tubes go in after PCR and before gel electrophoresis.
Alyssa Gao

 

Determining foliar N levels has three stages as well. First, leaves must be ground to dust using a coffee grinder and a mortar and pestle. Then, you wrap 5 mg of each sample into a tiny tin boat. Finally, you run these tinned samples through an Elementar, which burns them at an extremely high temperature. Technically, this analysis does not require rounds like my genetic work does. However, due to mistakes that I have made and technical issues with the Elementar, my samples have had to be re-tinned twice.

 

A coffee grinder, with its lid containing the ground leaf samples beside it.
Leaf samples after they have been ground my a coffee grinder.
Alyssa Gao

An open vial, a funnel, and a mortar and pestle.
Leaf samples being ground by a mortar and pestle, with its vial beside it and the funnel used to transfer the powder.
Alyssa Gao

A microbalance with a tin boat in the center.
Weighing a 5 mg sample of ground leaf in a tin boat using a microbalance.
Alyssa Gao

A 96 well-plate with folded tin samples in each well.
The majority of my tinned samples, ready to be run in the Elementar.
Alyssa Gao

My project has been further hindered by mistakes that I have made. Although several of my first attempts did not succeed due to mistakes I made with pipetting and with tinning, and other attempts have gone awry due to malfunctions with instruments, I have gained from my experiences and learned a great deal from my mistakes. I think the most important realization I have made during my time here is that research requires more than just persistence, attention to detail, and a willingness to ask questions. It requires optimism - the ability to remain positive and keep trying when things fail or when you make mistakes.


Even though research can be incredibly painful at times, it definitely pays off. Nothing is more satisfying than looking at the raw data you collected and knowing that it was a product of your hard work and determination, as well as the support of others. Even though roadblocks and mistakes are bound to happen, if you stay positive and keep trying, success in research will eventually happen too.

About the Author
Hi everyone! My name is Alyssa Gao, and I am one of the Undergraduate Research Fellows at the Morton Arboretum’s Center for Tree Science working with Dr. Chuck Cannon. I am a rising sophomore at Dartmouth College in Hanover, New Hampshire. This summer, I am researching the effects of bacterial leaf scorch on the leaves of Mongolian Oak specimens in the Morton Arboretum collections. Through this project, I hope to provide insight into the physiological responses of trees to disease. I am excited to contribute to the on-going research at The Morton Arboretum!