Rationale: To conduct PCR on the lysate

Procedure:

• The lysate was taken out of the shaking incubator.
• One mL of unfiltered lysate was taken out and placed in another tube.
• Then, it was placed in a centrifuge at 10,000 g for 10 minutes.
• After, the supernatant was taken out, carefully avoiding the pellet that formed at the bottom.
• The supernatant was then boiled for 10 minutes,
• After boiling, 2 µL of the enriched lysate ( in each of the three tubes) was added to the 2X Master Mix (12.5 µL of Taq polymerase and dNTP)
• 4 µL of primer was added to their respective tubes.
• 4.5 µL of ddH2O was added to each tube.

Results and Analysis:

Rather than putting 1 µL of enriched lysate solution, 2 µL were put into the primers.

This time, a clear tack was used rather than a dyed one. When doing gel electrophoresis, the dye must be added to the primer.

Conclusion:

Because there were no phages on the plates, another approach to check for phages was taken. The first step to conduct PCR was to boil the enriched lysate was 95 degrees Fahrenheit. Then, 1 µL of the enriched lysate was added to the 2X Master Mix along with 6.5 µL of ddH2O. Primer was added to their respective tube. This was conducted three times, adding each component in equal volumes.

Future Plans:

In the future (10/31/18), gel electrophoresis will be performed using the lysate samples treated with primer.