Daphne Simo

02/07/19

I. TITLE

DNA Extraction

II. RATIONALE/PURPOSE

The purpose of this lab was to perform extract DNA from our soil samples utilizing metagenomic methods in preparation to purify the DNA. Furthermore, we determined the soil texture of our samples as data to answer our overraching research question.

III. MATERIALS

Dissecting Microscope

Petri Plates

Soil sample in Falcon tube

Centrifuge

Centrifuge tube

Pipette

Chelex

Heat block

DNA extraction buffer

Silica beads

Mortar and pestle

Activated charcoal

Eppendorf tube

Leaf

IV. PROCEDURE

Soil Texture

1. Use a ruler to measure the overall amount of soil in sample in centimeters
2. Measure the amount of sand, silt, and clay present in the overall soil
3. Use the measurements of each type of texture present and divide by the overall amount of soil in the sample to get the percentage of each type
4. Use a Soil Triangle and Soil Type Calculator to determine the soil texture

DNA Extraction

1. Weigh out 1 gram of the selected soil sample and 1 gram of glass silica beads into a dish on a balance
2. Place the combined soil sample and silica beads into a mortar and begin to grind the components together using a pestle for about 5 minutes
3. Add 2mL of DNA extraction buffer into the mixture
4. Add 10mg of powdered activated charcoal into the mixture
5. Combine the mixture together by pipetting it multiple times
6. Transfer the mixture into an Eppendorf tube
7. Incubate the mixture in the Eppendorf tube at 65°C for 10 minutes in a water bath
8. Centrifuge the tube at 12000g for 5 minutes at 4°C
9. Take out 500μL of supernatant to a fresh microfuge tube

Tree Metadata

1. Determine the Genus species of your selected tree
2. Sketch a detailed drawing of your leaf
3. Use the “Figure of Leaves” to describe the leaf in as much detail
4. Put leaf under dissecting microscope to closely observe the leaf for other intricate features

V. DATA/OBSERVATIONS

Soil Texture

Total of sand, silt, and clay: 1.85 cm

Sand: 0.7 cm (0.7/1.85) = (29.73%)

Silt: 0.6 cm  (.85/1.85) = (32.43%)

Clay: 0.55 (0.55/1.85) = (4.76%)

Texture: Clay loam

Image: Falcon tube containing soil sample which was used to measure the soil texture

DNA Extraction

Images: Using a pestle to combine the soil sample and silica beads together (left). Using a micropipettor tip to mix the DNA extraction buffer, activated charcoal, soil sample, and silica beads (right)

Tree Metadata

Genus species: Quercus imbricaria

Tree name: Shingle Oak

Observations: The shape and arrangement best resembled an elliptical and linear leaf. It was oval shaped with a broad point on the top. The leaf was very elongated with parallel margins. Then venation was reticulate with small veins interconnecting. Lastly, the margins were very even and smooth. We determined the Genus species by using the “Identify by Leaf” test.

Image: Leaf sample taken from selected tree (left), Sketch of leaf sample (right)

VI. STORAGE 

The soil samples were stored away per instructions from our professor for future use. On the other hand, the used pipettes and centrifuge tubes were discarded. Microscopes were unplugged and covered and lab tables were thoroughly cleaned off. Our DNA sample was labeled and stored properly.

VII. CONCLUSION

I really enjoyed performing this experiment as it allowed us to delve firsthand into DNA extraction–something many of us may not have done before. It encouraged me to be very meticulous when it came to performing the protocol, as the DNA extraction was a vital aspect of our research in lab. While doing the extraction procedure, we utilized activated charcoal and DNA extraction buffer. The activated charcoal enabled us to extract the impurities , while the DNA extraction buffer chelated the cofactors within our sample. After performing the experiment, it is our hope to find have done it successfully so we can continue the process by purifying the DNA. On the other hand, it was interesting having to draw our leaves in lab. I figured it means that researchers should be multi-faceted in order to perform a vareity of tasks which will consequently help the experiment as a whole.

VIII. FUTURE STEPS

Moving forward, I hope to have done the DNA extraction properly so we can move forward with our research. I hope to also solidify in my mind how each other the steps in the DNA extraction process work as a whole.