Megan Tran

2/15/19

Objective:

The objective purpose of today’s lab was to take our DNA extracts from last week’s lab and purify them and to make an agarose gel for gel electrophoresis to sort and measure DNA strands in our samples.

Procedure:

Making the Agarose Gel

1. Add 4 mL of stock solution and 36 mL of D.I. water to a conical tube.
2. Add 0.4 g of agar powder to an Erlenmeyer flask.
3. Add both the stock solution + D.I. water to the Erlenmeyer flask and swirl until mixed well.
4. Microwave the solution for one minute in the flask.
5. Remove the solution from the microwave and let it cool.
6. Once cooled, add 2 μl of Et Br to the solution and swirl until the solution appears clear.
7. Assemble a gel electrophoresis mold.
8. Pour the solution into the mold and wait for 15 minutes until the solution is cooled and set.
9. After cooled, fill the rest of the mold with 1xTAE buffer solution to prevent it from drying out.
10. Place the entire mold into a plastic bag and store in the refrigerator.

DNA Purification

1. Add sterile water to the crude soil DNA extract until it reaches 1 mL, then add the 1 mL to a 15 mL tube.
2. Add 2 mL of warm DNA resin. Invert the tube several times to ensure that it is mixed before pipetting 2 mL to the tube.
3. Set up a column bottom on a syringe barrel and set on the vacuum filtration manifold.
4. Add half of the DNA resin to the column and turn on the vacuum. Once all of the liquid is pulled through the column, add the rest of the sample.
5. Wash the column by gradually adding 2 mL of 80% isopropanol, using the vacuum to pull it through. Repeat two more times.
6. Remove the column from the barrel and put the column in a clean 1.5 mL tube. Spin at 8000 x g for 5 minutes.
7. Remove the column out of the 1.5 mL tubes and put it in the 80 degrees Celsius heat block for 1 minute.
8. Put the column in a new 1. 5 mL tube and apply 50 μl of D.I. water to 80 degrees Celsius.
9. Incubate for 1 minute.
10. Spin at 8000 x g for 1 minute.
11. Measure the concentration of DNA for each elution on the NanoDrop and run 2μl on a gel.

Storage:

The agarose gel molds were placed and sealed in a plastic bag and stored in the refrigerator.

Conclusion:

Through today’s lab, we learned how to extract DNA from our sample with a fairly inexpensive method, and we will use this information from the DNA to contribute to our overall goal of developing a working protocol for determining the diversity of ciliates in the soil. Learning how to perform a gel electrophoresis and DNA purification procedure was a fairly enjoyable process and I am now prepared to do these procedures in the future.