Rationale

Phage presence will be tested on the enriched isolation produced on 10/15/18 with soil sample E using PCR.

Procedure

• Established an aseptic zone.
• 8.5 mL of enriched isolation was aliquotted into a tube and centrifuged at 5000g for 5 minutes.
• 1 mL of the centrifuged enriched isolation was put into a microcentrifuge tube and was heated for 10 minutes.
• 4 µL of primer mix 1, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 1 along with 12.5 µL of TAQ polymerase.
• 4 µL of primer mix 2, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 2 along with 12.5 µL of TAQ polymerase.
• 4 µL of primer mix 3, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 3 along with 12.5 µL of TAQ polymerase.
• 12.5 µL of TAQ polymerase, 1 µL of “phage DNA”, 4 µL of primer mix 1, and 7.5 µL of D.I. water were placed into microcentrifuge tube 4 to make a positive control.
• The 4 tubes were then cycled through PCR.

Observations

D.I. water was used in the tubes rather than D.D.I water, which may have an effect on the results of the experiment.

Conclusions/Next Steps

Using the PCR results, gel electrophoresis will be run on Monday to test for phage presence in the sample.