Rationale: Plaque Assay plates results were negative and control plate was contaminated. Continued by performing a PCR test.  

Procedure: 

1. Aseptic zone was created using an ethanol burner and cleaning table with ethanol and Cidecon.  
2. Obtained enriched lysate from fridge and transferred 4 mL of enriched lysate with a mass of 10.99 g into 15 mL tube to be centrifuged. 
3. Centrifuged the enriched lysate for 5 minutes at 3,000 g.  
4. Transferred 1 mL of centrifuged enriched sample into small tube in an aseptic zone since lysate was previously filtered, using a micropipette.  
5. Returned the original lysate to the fridge.  
6. Boiled the lysate for about 10 minutes.  
7. Obtained 4 tubes containing 12.5 uL of Taq Polymerase, three for primers, and one for a control.  
8. Pipetted 1 uL of enriched lysate of 2 soil samples into each of the tubes labeled for the three primers.  
9. Added 6.5 uL of DI water into the three primer tubes, and 8.5 uL in the control tube.  
10. Added primer 1, primer 2, and primer 3 to respective tubes, and primer 1 to the control tube.  
11. Stored to be used next class for a gel electrophoresis.

Observations/Results: 

The plaque assay plate returned negative and had no contamination and therefore no plaques were present. The control plate was contaminated.  

Individual Plate – no contamination or plaques

Control Plate – contaminated

PCR : 

For PCR used DI water instead of a more purified water, which could alter the results slightly.  

Next steps: 

Next class we will continue with the PCR test, by running a gel electrophoresis to see if the soil samples being tested has DNA, and therefore phage.