Daphne Simo

02/15/19

I. TITLE

DNA Extraction Part 2

II. RATIONALE/PURPOSE

The purpose of this lab was to perform purification of the DNA from our soil samples and create agarose gel in preparation for gel electrophoresis.

III. MATERIALS

Crude soil DNA

Deionized water

DNA resin

15mL tube

Syringe barrel

Column bottom

Vacuum filtration manifold

2mL 80% isopropanol

Centrifuge

Heat block

Tris-EDTA Acetic Acid (TAE)

Erlenmeyer flask

Weighing paper

Microwave

Ethidium bromide

IV. PROCEDURE

DNA Purification

1. Add 1mL of sterile water into the crude soil DNA if not up to 1mL and place this into a 15mL tube for mixing.
2. Add 2mL of 37°C DNA resin and mix the solution by inverting the tube.
3. Set up a column bottom on a syringe barrel and place it on a vacuum filtration manifold.
4. Turn on the vacuum and add half of the DNA and resin to column.
5. Gradually add the DNA resin solution through the column after the first set of the sample has gone through the column.
6. Clean the column by adding 2mL of 80% isopropanol via the vacuum with the same process used to place the DNA resin solution.
   • Repeat the above process for a total of three times.
7. Remove column from the barrel and place the column into a clean 1.5mL tube.
8. Place the tube into a centrifuge and spin at 8000 x g for 5 minutes
9. Take the column out of the 1.5mL tube and place into a heat block for 30 seconds at 80°C, making sure not to exceed a minute.
10. Put column in a new 1.5mL tube and add 50mL of deionized water directly into the column heated to 80°C .
11. Incubate the column for 1 minute.
12. Spin at 8000 x g for 1 minute again.
13. Label your groups tube.
14. Measure the concentration of the DNA for each solution on the NanoDrop and run 2μL on a gel with DNA MAss Standards to check the purification and concentration

Agarose Gel Preparation

1. Weigh out 0.4 grams of agarose powder into a small Erlenmeyer flask using a spatula and weighing paper on a balance.
2. Put deionized water into the agarose powder and mix the two components together.
3. Heat the solution in the microwave until the solution is clear and small bubbles rise from the bottom when swirled.
4. Allow the solution to cool for about 5-6 minutes.
5. Add 2μL ethidum bromide into the solution once cooled.
6. Pour the cooled agarose solution into a gel electrophoresis box, making sure no bubbles are present.
7. Allow the agarose solution to solidify for about 30 minutes

V. DATA/OBSERVATIONS

No data collected for this lab.

VI. STORAGE 

The gel electrophoresis box was stored away by our lab assistants properly. The materials used to create the gel electrophoresis and agarose gel (i.e. were placed into its correct location.

VII. CONCLUSION

Performing this lab allowed me to understand the importance of being very thorough when doing research work. Furthermore, it introduced me to the process of gel electrophoresis and heightened my curiosity of how it works.

VIII. FUTURE STEPS

Moving forward, I hope to have done the DNA purification accurately in order to move on to the next step to answer our overall research question. Likewise, I hope to research more about gel electrophoresis to enhance my knowledge of it.