Date: Wednesday, October 24th, 2018

Title: Gel Electrophoresis

Rationale: The purpose of today’s lab is to perform gel electrophoresis in order to test samples against a DNA ladder to determine whether or not the sample contains phage DNA.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. 35 mL of TBE buffer was added to a flask.
2. .72 g agarose was added to the flask with TBE buffer.
3. The flask was placed in a microwave and cooked in intervals until the solution was clear.
4. The flask was allowed to sit until it was cool enough to hold with a bare hand comfortably.
5. 1.7 microliters of Ethidium Bromide (EtBr) was added to the flask.
6. The solution in the flask was poured into a tray and allowed to harden
7. A thin layer of TBE was added over the hardened tray so that the comb and dams could be removed without tearing the gel.
8. TBE buffer was added to the gel electrophoresis tray until it covered the gel section.
9. Four wells were used:
   1. The first well was filled with 5 microliters of DNA ladder which would serve as a match to possible DNA.
   2. The second well was filled with 10 microliters of the small tube with PM 1. 2.5 microliters of dye was added to the tube before the contents were added to the well in order to weigh down the DNA so it did not float from the well.
   3. The third well was filled with 10 microliters of the small tube with PM 2.
   4. The fourth well was filled with 10 microliters of the small tube with PM 3.
10. The gel was connected to a power source and allowed to run for 45 minutes. The gel was imaged and yielded negative results.

Observations:

The results from the gel electrophoresis did not match the expected results based on the ladder. This means that the sample in all likelihood did not contain phage DNA.

The compound EtBr is a carcinogen and must be handled carefully. Gloves were used when in contact with EtBr.

Results: This experiment yielded negative results. The PCR and gel electrophoresis did not match the DNA ladder and did not contain phage DNA.

Next Step: The next step is to either collect more soil or adopt from another group.