Bassam Ballout

Lab Notebook 5

Thursday, February 14th, 2019

Aim:

The aim of this lab was to purify our DNA extraction and make agarose gels to later use in gel electrophoresis. We also prepared for DNA analysis using the gels to aid in determining a possible match of DNA in the soil.

Materials:

• crude soil DNA
• 15 mL falcon tubes
• DNA Resin
• column and syringe
• Vacuum filtration
• isopropanol
• centrifuge
• heat block
• Distilled water
• TAE stock solution
• Agarose gel
• Erlenmeyer Flask
• Microwave
• Ethidium Bromide
• Fridge

Procedure:

DNA Purification:

1. Obtain the crude soil DNA
2. Place it into a 15 mL falcon tube and make sure it contains at least 1 mL of DNA.
3. Then add 2 mL of DNA resin and mix by flipping.
4. Once that is done set up a column and syringe on the vacuum filtration manifold.
5. Add DNA resin to the column.
6. Add isopropanol in .5 mL increments until 2 mL of 80% isopropanol is added in total.
7. Remove column from barrel and place into a clean 1.5 mL tube.
8. Centrifuge the tube for 5 minutes to get rid of rest of isopropanol.
9. Place the column in the heat block for 30 seconds.
10. Add 50 µl of distilled water to the tube and heat to 80 degrees Celsius in heat block for a minute.
11. Then centrifuge the tube for 1 minute.
12. Label tube and store your tube.

Agarose Gel:

1. Place 0.4 g of Agarose, which was weighed on a balance, into an Erlenmeyer flask.
2. Add the 1 x TAE solution to the flask and swirl properly.
3. Microwave the solution until the Agarose is fully dissolved and let it cool down.
4. Once the flask is cool enough, add 2 µg of ethidium bromide and stir thoroughly.
5. Place the comb inside the gel electrophoresis with the blue part of the comb facing the side away from the space in the middle.
6. Once done stirring, pour the solution into the gel cast and wait for the gel to begin solidifying.
7. Once the gel has solidified you may remove the comb from the gel and set it aside.
8. Label the gel mold and store the gel mold in the refrigerator.

Results:

No results were recorded during this lab, however we will be using the procedures carried out in this lab in the next lab to properly obtain quantitative data.

Conclusion:

Our group was able to properly form the gel mold inside the gel electrophoresis and complete the DNA purification. In the next next lab we will use the mold and DNA to see what our soil DNA bands looks like after we carry out the entire electrophoresis. We have also eliminated all impurities and therefore should obtain clear and valid data throughout our next coming labs.

Future Steps:

Using the gel mold and soil DNA extraction, we will continue to move towards our goal of metabarcoding the ciliates to obtain their DNA to further understand the life of a ciliate and how they function using gel electrophoresis.