Date: Monday, October 22nd, 2018

Title: PCR Soil Sample C

Rationale: The purpose of today’s lab is to perform PCR to replicate and search for phage DNA in the soil sample.

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. The enriched lysate from soil sample C was centrifuged at 3000 gs for 10 minutes.
2. 1 mL of the centrifuged lysate was transferred to a microcentrifuge tube and boiled to release possible phage DNA from capsids.
3. 6 small tubes were set up. Three were used as a negative control and three used the lysate from soil sample C as well as a partner’s lysate in order to test for phage DNA. The tubes were set up the following way:
   1. 12.5 microliters of Taq Polymerase was added to each of the 6 tubes.
   2. 4 microliters of primer mix 1, 2, and 3 were added to the negative control tubes and the experimental tubes marked with 1, 2, and 3 respectively.
   3. 6.5 microliters of ddH2O were added to each of the 6 tubes.
   4. 2 microliters of lysate (1 microliter from the soil sample C lysate and one microliter from a partner’s lysate) were added to each of the 3 experimental tubes.
   5. An extra 2 microliters of ddH2O were added to each of the 3 negative control tubes.
4. These tubes were all put into a heat cycle in order to let the primers match with possible phage DNA.

Observations: The experimental tubes marked for PM (primer mix) 2 and 3 were green while the tube marked for PM 1 was clear. This is because a different material was used in the tube. For gel electrophoresis, a dye will have to be added to the first tube in order to weigh DNA down.

Results: This experiment yielded three experimental tubes that can be tested with gel electrophoresis to search for the presence of phage DNA. This experiment also yielded three negative control tubes that can be tested against to make sure there isn’t contamination or that a procedure was done incorrectly since these tubes should result in strictly negative results.

Next Step: The next step is to use gel electrophoresis in order to check for phage DNA. If this yields negative results, the next step is to find new soil or to adopt phage/soil from another group.