10/24/18 Soil Washing and Enrichment of Soil with Known Phage

Objective:

The goal of this procedure is to test the efficacy of PCR and Gel Electrophoresis. The way I will be testing this is by creating a newly enriched lysate using soil that already tested positive for phage in the previous testing. The purpose behind this is to test if different ways of cleaning the enriched lysate will affect the results of PCR. I will do this in future labs by using a syringe filter to filter half of the enriched lysate I create, and using chloroform to clean the other half, and then running PCR and Gel Electrophoresis on each sample.

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 Soil Washing:

• Syringe filter
• .5 ml Arthrobacter
• 50 ml conical vial
• 15 ml conical vial
• LB Broth
• refrigerator
• Incubator
• Centrifuge
• 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 soil was washed and enriched according to the following procedure:

1. Gather previously positive soil
2. ~2.5 ml of soil was added into a 15 ml conical vial
3. 11 ml of LB broth was added
4. The vial was vortexed for 15 minutes
5. The vial was then centrifuged for 10 minutes
6. ~ 1.5 ml were pipetted into a fresh 15 ml vial for a direct lysate and placed in the fridge
7. ~9 ml of lysate were pipetted into a fresh 15 ml vial
8. .5 ml of Arthrobacter was added to the 15 ml conical and put in the incubator

Results:

The results of this procedure will not be immediately clear because I am not testing for phage presence. After I do the two different procedures to clean the enriched lysate and then run PCR and Gel Electrophoresis I will report on my results. Aside from that, I can say that this appeared to go well and an enriched lysate was created.

Analysis:

PCR works by using polymerases to make many copies of specific strands 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 allows people to determine whether or not there is phage DNA in an enriched lysate. Gel Electrophoresis works by using electricity to separate different strands/fragments of DNA in order to analyze the patterns that ensue.  When electricity is applied, DNA moves toward the oppositely charged end of the gel tray and brings the DNA dye with it. The question of my next few procedures seek to address is whether or not these procedures accurately indicate DNA presence and if they can be affected by different prep methods.

Future:

For my next three lab procedures I will be utilizing two different cleaning methods on my enriched lysate, then running PCR, and then doing Gel Electrophoresis.