Objective

The objective of this lab was to purify our eDNA sample that we extracted the previous week. In addition to removing all of the debris from the eDNA sample, we also made an agarose gel that we can use for gel electrophoresis next week.

Purpose

The purpose of purifying the eDNA was to remove any microscopic debris that we extracted along with the genetic material. The debris could cause issues with the gel electrophoresis, so we needed a pure eDNA sample. The purpose of making the gel was simply that it is the medium we will require to run a gel electrophoresis next week.

Procedure

DNA Purification

• Add deionized (DI) water to the crude soil DNA extract up to the 1mL mark. Put this 1mL of solution into a clean 15mL tube appropriate for mixing.
• Add 2mL of warm (37 degrees Celsius) DNA resin from the Wizard PCR Prep DNA Purification kit.
• Set up a column and syringe barrel on a vaccuum filtration manifold.
• Add half of the mixture in your 15mL tube to the barrel. Once all the liquid has been pulleed through, add the other half.
• Wash the column by adding 2mL of 80% isopropanol to the barrel. Add 1mL at a time up to 6mL total (3 total washes, 2 mL per wash)
• Remove the column from the syringe barrel and place in a clean 1.5 mL centrifuge tube. Spin at 8000 x g (or 8000 rcf) for 5 minutes.
• Take the column out of the centrifuge tube, disopse of the tube, and place the column in a new 1.5 mL tube, heating it for 30-60 seconds in an 80 degrees Celsius heat block.
• Add 50 microliters of DI water to the column and let it incubate for 1 minute.
• After the minute is up, spin again at 8000 x g for 1 minute.
• Take the tube out, and dispose of the column.
• Label the tube and place it in the freezer for storage.

Data

No data was recorded in this lab; it was mostly just setting the stage for the gel electrophoresis we will run next week.

Conclusion

This lab was definitely one of the longest. We all had to figure out the procedure and wait for our processes to be done, but it was worth all the work in the end. After all, purifying the DNA is an essential step. While my partner Anthony and I were purifying our eDNA, my other partner Emely was preparing the agarose gel we are going to use next week.

Storage

Once purified, our DNA samples were put in the freezer to prevent them from denaturing. I ensured that our station was neat and all of our trash was disposed of accordingly.