22 October 2018 ✷ Soil 5 gel electrophoresis

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was tested using gel electrophoresis in order to look for presence of phage DNA.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• 0.8 g of agarose powder was mixed with 40 mL TBE (buffer) and microwaved. Ethidium Bromide was added and the solution was poured into a gel apparatus and a 8-prong comb was added and the gel was allowed to harden. It was then transferred to an electrophoresis tray and filled with TBE.
• The first well was used for other group members to practice. The remaining wells were as follows:
  • Well 2: “(: 1” LL Sam’s Oak DNA + Other group’s DNA + primer 1
  • Well 3: “(: 2” LL Sam’s Oak DNA + other group’s DNA + primer 2
  • Well 4: “(: 3” LL Sam’s Oak DNA + other group’s DNA + primer 3
  • Well 5: DNA Ladder
  • Well 6: “★1” DNA from 2 other groups + primer 1
  • Well 7: “★2” DNA from 2 other groups + primer 2
  • Well 8: “★3” DNA from 2 other groups + primer 3
• The gel electrophoresis was run for roughly 30 minutes at 100 V and analyzed with UV.

observations, results, data

The gel, when analyzed with the UV scanner, was negative for phage DNA. The controls all worked, so the test was effective and the soil tested did not contain phage.

The other two samples collected on the same day, from the basketball hoop and electric pole, were also negative. The use of DI water instead of DD water did not have an adverse effect on the gel.

The gel pictured above “glowed” more than other groups, but this is likely because it ran for less time than the other groups’ gels and there was a higher concentration of dye in one spot instead of being dispersed throughout the entirety of the gel.

interpretations, conclusion, next steps

It is possible that the chosen variable for group 6, red oaks, is a negative one, meaning that the scientific question “is there a correlation between the presence of phage in white vs red oaks?” is being answered with, “there are no phages near red oaks” because every sample from red oaks has been negative for phage. It is still somewhat premature to make this conclusion, but the data has not proved this statement incorrect as of yet.

In the coming days, more soil will be collected and enriched. Then, it will be run through PCR and gel electrophoresis.