Lab 4: DNA Extraction – February 7th, 2019

Objective

The main purpose of this weeks lab is to finalize our soil metadata calculations, specifically soil texture, water composition, and tree classification, as well as start the process of DNA extraction from the ciliate cultures.

Rationale

Our experimental question for this portion of the unit is: What is the biodiversity of Baylor trees? We are addressing the experimental question by collecting soil metadata as well as extracting DNA from the soil ciliates. This week, the metadata that we are calculating is the soil texture, soil percent water content. We will also determine the tree species.

Methods

Soil Texture

The vortexed sample in the falcon tube from last week (Methods in the last lab) was left to settle into layers, and the texture of the soil was calculated by measuring the width of each layer of clay, sand, and silt. The measurements were then used to place a point on the soil texture triangle, revealing the soil texture.

Soil Water Composition

The measured soil in the petri dish from last lab (methods in last weeks lab) was left to dry for a week. The soil was remeasured and the water composition was calculated by a simple formula that includes the weight of the dish, the weight of the wet soil, and the weight of the dry soil.

DNA Extraction (Chelex Extraction Protocol)

500 microliters of dense (cloudy) ciliate culture were placed in a microcentrifuge tube. The ciliates were taken from the culture in well A4. The microcentrifuge tube was then centrifuged at 6000g for 5 minutes. The supernatant was discarded, and if the pellet was smaller than a few millimeters, then another 500 microliters of culture were added and recentrifuged. After the pellet was large enough, 200 microliters of 5% Chelex 100 was pipetted into the centrifuge tube and vortexed for 1 minute. The tip of the micropipette was cut off in order to let the Chelex pass through the tip. 2 microliters of protease were then added to the tube and the solution was placed into a freezer to pause the procedure. The rest of the DNA extraction will continue next week.

Results

Soil Texture

The individual layers of clay, silt, and sand were calculated to be:

Clay: 8% Silt: 29% Sand: 63%

According to the soil texture triangle, this soil is classified as Sany Loam.

Soil Water Composition

The following masses were used to calculate the soil water composition.

Wet Soil: 17.9 grams Dry Soil: 14.7 grams

Water percent: 18%

DNA Extraction

This process is half-way finished, results will be finalized in the future.

Conclusion

I think that the calculations for the soil metadata were fairly easy to follow through with, they required basic math skills. The DNA extraction procedure was entirely new and very interesting. We were asked to research the prices of the Chelex, it is very expensive. Each group was asked to decide between extracting DNA from the soil sample directly or extracting from the ciliate cultures made last week. Since my group was able to successfully culture a good amount of ciliates, we decided to go ahead and extract DNA from that culture.

Future Goals

I am ready to continue with the DNA extraction because it is very interesting to me, unfortunately, we had to punctuate the procedure this week by storing it in the fridge until the next lab meeting. I hope the DNA extraction will successfully yield results that can be interpreted toward our experimental question.

Storage

The extraction solutions were placed into a freezer, progress will continue next week. The dry soil was returned to the back of the room.