Abel Thomas

2/14/19

Objective: Today’s lab objective was to assemble all the necessary components we would need to sequence and observe the DNA we collected from our soil.

Materials:

Ethidium bromide

Resin

125ml Erlenmeyer flask

falcon tubes

15 mL test tubes

agarose gel

column

syringe barrel

80% isopropanol

gel mold

comb

Procedure:

DNA Extraction:

1. Make sure the falcon tube contains 1 mL of DNA, if not, add D.I water
2. Add to a 15 mL test tube
3. Add 2 mL of resin into solution and mix by inverting it multiple times
4. Set up a column and syringe barrel on the vacuum filtration manifold
5. Add half  of the solution to the column until it gets sucked through, then put in the other half
6. Wash the column by adding 2 mL of 80% isopropanol and use the vacuum to pull it through
7. Repeat the washing step over again so it rinses clearly
8. Remove the column from the barrel and put it into a clean 1.5 mL tube
9. Spin the column in a centrifuge for 5 minutes to rid of the rest of the isopropanol
10. Put the column into the heat block for 30 seconds to a minute.
11. Put the column in a new 1.5 mL tube and add 50 uL of D.I. water heated to 80 degrees Celsius directly to the column.
12. Incubate for a minute and then spin again in the centrifuge for a minute

Agarose Gel:

1. Measure .4 grams of the agarose powder on a balance
2. Put it into a 50 mL test tube and add D.I. water so it would reach the 40 mL point
3. Shake the tube by inverting it
4. Pour the solution into a 125 mL Erlenmeyer flask
5. Microwave and heat the solution for a minute or until the agarose dissolved
6. Let the solution cool and add 1omg/mL Ethidium Bromide and swirl the final solution together
7. Place the solution into the gel electrophoresis mold
8. Place comb correctly by making sure blue is facing away from the major area of the mold
9. Let the solution cool until it is solidified
10. Remove the comb

Storage:

We made sure we wiped down the table and disposed of all the unnecessary materials. We also made sure to wash and rinse all of the beakers that were used.

Conclusion/Future Goals:

We were able to successfully set up our agarose mold and make sure we isolated our DNA so it would be ready for electrophoresis next week. Our future goal is to use what we used this week and our prior knowledge to use the gel electrophoresis next week to help show which DNA is the best to be sequenced.