Repeat Enrichment due to bad LB Broth

10/1/2018

Objectives: Repeat Enrichment Procedure to separate a lysate, collect metadata

Rationale: Must have a lysate to isolate phage, if present, and must have metadata to better analyze potential patterns in phages.

Procedure:

• Isolated 2 mL of the same soil from last lab
• Added 10 mL LB Broth
• Shook 15 Min
• Centrifuged tube for 10 min at 5000 rpm

When centrifuging, completed metadata

• Massed dry weigh boat
• Found water to make up 40.2% mass
• Determined soil makeup to be comfortably clay

Removed mixture from Centrifuge

• Filtered through vacuum apparatus
• Left lysate in shaking incubator

Cleaned lab station and left.

Nothing to analyze or interpret.

Future Plans: Complete a Plaque Assay on new Lysate.

Plaque Assay 3

10/5/18

Results from last lab: Last Lab I was busy and couldn’t make it in for long enough to call it an actual day. I moved the lysate from the shaking incubator to the freezer, and left.

Objectives: Complete Plaque Assay on Lysate 3

Rationale: In order to check for the presence of phages, a spot test or plaque assay needs to be performed. Most groups have found a plaque assay to be more effective and valuable time wise.

Procedure:

• Filtered lysate into micropipette tube through a 22 nm filter
• Added 2 mL LB Broth, .5 mL Arthrobacter, 22.5 microliters Calcium Chloride, and 2.5 mL 2X Top Agar to 15mL conical tube
• Added mixture to plate to solidify
• Found that Arthro had condensed and gotten stringy- rather than throwing it out, it was labeled and added to the incubator
• Added 2 mL LB Broth 2.5 mL 2X Top Agar to a 15mL conical tube
• Added 2 mL LB Broth, .5 mL Arthrobacter, 22.5 microliters Calcium Chloride, and 2.5 mL 2X Top Agar to 15mL conical tube
• Arthro dispersed properly this time, so both the control and plaque assay plates sat for 15 minutes, and were placed in the incubator

Analysis and Interpretation: Verify date of arthro before use in the future. Investigate how the arthro turns out next lab period.

Future Plans: Analyze Plaque Assay for the presence of phages, and continue inputting soil metadata into the survey on canvas.