October
29
10.29.18 PCR for Claire and Lucy
10.29.18 PCR for Claire and Lucy
Rationale: Since no phage had been obtained from any samples previously run, it was found most pertinent to help with other samples that had more promise of developing a high titer lysate. Therefore, a PCR test was run for samples from Claire and Lucy to help verify the PCR procedure was working properly.
Procedure:
- Aseptic zone established
- Obtained enriched lysate from LIP Soil Sample C
- 1mL enriched lysate from LIP Soil Sample C was added to a microcentrifuge tube
- Microcentrifuge tube placed in 37°C container for 10 minutes to boil
- Obtained 3 tubes with 12.5μL of Taq Polymerase in them. Tops labeled star 1, star 2, star 3.
- Added 4.5μL DDI water to each tube
- Added 4μL Primer 1, Primer 2, and Primer 3 to corresponding tubes
- Added 2μL of boiled enriched lysate from LIP Soil Sample C and Claire’s sample.
- Thermocycled all tubes.
- Cleaned and tidied bench.
Results/Observations:
- No new results regarding soil samples were obtained today after the negative result found on Wednesday 10/24.
- The enriched sample that was used was slightly pink in color, which is different from the normal pale yellow.
- Volume of sample added to the PCR tubes appeared to be slightly varied rather than a consistent 2µL. Some drops struggled to fully reach the bottom of the tubes.
Conclusions/Next Steps:
- The PCR tests run today will serve to show if the PCR procedure is working correctly. Since there have been no positive results using the PCR procedure (other than some positive controls), it was found necessary to run a sample that is known to have phage that was found in the class. After a gel electrophoresis is run on Wednesday 10/31, it will be known whether or not the sample is working correctly.