10/17/18 PCR on Soil Sample #3

Objective:

The goal of this procedure is to prep for and begin PCR on the enriched lysate created in the last lab. PCR will help us determine if there is phage DNA present, which will determine whether or not we do future testing on soil sample #3.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials For PCR:

• 15 ml conical vial
• PCR Machine
• DI Water
• TAQ Polymerase
• Centrifuge
• microcentrifuge tube
• pipette
• Test tube stand

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table
2. 70% Ethanol was also applied

The enriched lysate was prepped for PCR:

1. The previously created enriched lysate was spun in a centrifuge for 5 minutes to pellet the arthro
2. ~1 ml of lysate was transferred to a  Microcentrifuge tube
3.  The tube was boiled to release phage DNA

PCR tubes were created:

1. 4 PCR tubes were created according to the following recipe:
2. *note DI water instead of DDI water was used on accident*
3. the tubes were placed in the PCR machine until next lab

Results:

The majority of the information that will be available from these procedures will not be visible until the next lab, so these results will be updated on Monday when the results of PCR will be visible and more testing can be conducted. However,  it seems as though PCR testing went well with the exception of the mistake in water.

Analysis:

PCR works by using polymerases to make many copies of specific stands of DNA  so that it is easier to analyze. Using PCR allows researchers to take small samples and amplify the genetic contents in order to conserve resources and determine what is present before future testing commences.  In this lab, using PCR will allow me and my partner to determine whether or not there is phage DNA  in our enriched lysates before we take the time to run spot tests and plaque assays. If everything went according to plan, and the PCR works correctly, then this will likely save us a great deal of time.   It is possible that the additional ions found in DI water as opposed to double distilled water may affect the results of the testing, and this will be something important to note if we receive weird results when we do gel electrophoresis.

Future:

This procedure might have been derailed because I accidentally used DI water instead of double DI water. If this interferes with the PCR then I will have to conduct PCR again, if it does not affect PCR, then I will be using gel electrophoresis to search for phage DNA.