SEA Bears Day 16
17 October 2018 ✷ Soil 5 amplification
Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was amplified using PCR to determine if the sample was worth spot testing for phage.
Procedure
- The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
- 4 mL of enriched lysate was transferred to a 15 mL conical vial and centrifuged at 3000 g for 5 minutes. 1 mL of the separated supernatant was placed in a microcentrifuge tube and heated. 1 µL of soil sample 5’s “phage DNA” was added to 3 PCR tubes. Volumes can be seen below.
| tube 1 | tube 2 | tube 3 | negative control 1 | |
| DNA 1 (soil 5) | 1 µL | 1 µL | 1 µL | – |
| DNA 2 | 1 µL | 1 µL | 1 µL | – |
| DI water | 6.5 L | 6.5 µL | 6.5 µL | 8.5 µL |
| TAQ Polymerase | 12.5 µL | 12.5 µL | 12.5 µL | 12.5 µL |
| primer 1 | 4 µL | – | – | 4 µL |
| primer 2 | – | 4 µL | – | – |
| primer 3 | – | – | 4 µL | – |
- The DNA samples were then cycled through PCR and stored until Monday.
Observations, results, data
The water used in the PCR tubes was DI water instead of doubly purified water, which shouldn’t ruin the PCR but may make some impact.
Metadata from Monday was finalized and recorded in Monday’s post.
interpretations, conclusion, next steps
The primers in each of the tubes should bind to any DNA known to an arthrobacterphage, meaning the use of a gel electrophoresis should result in the visual confirmation of phage DNA in the samples. If a sample is positive, it means that one of the DNA samples within it was positive, so a spot test or plaque assay would be conducted to determine which was positive.
Next lab, a gel electrophoresis will be run to test for phage DNA.