Notebook Week 11 – Pre-lab Name: Lily Hewitt Date: 04/05/2022 Lab Section #11 PRE-LAB (from materials posted on Canvas the Sunday before lab) 1. Title: Pure Phage Enrichment: Preparing a High-Titer Lysate 2. Purpose/Objective: To feed the phage the host cells to amplify the phage concentration and creating what is called a High-Titer Lysate or HTL. 3. Materials: Isolated Plaque from most recent and final plaque streak plate, .5ml of foliorum, 25 ml of medium, sterile wooden applicator, syringe filter, microfuge tubes, phage buffer, micropipette and micropipette tips 4. Procedure: So, for this procedure I will be conducting the part after the enrichment. Therefore, I will retrieve a 0.22 um syringe filter and use a 3 ml syringe to pull my enrichment lysate. Then I will attach it to the 0.22 um filter on the syringe and dispense the liquid through the filter into 3 microfuge tubes. Then I will be performed the 10 fold serial dilutions by arranging 10 microcentrifuge tubes in a test tube rack and added 90 ul of phage buffer to each tube. Then I will add 10 ul of the undiluted HTL to the tubes and mix. After using a fresh sterile tip, I will add 10ul of the -1 and -2 tube and finger vortex well. Then I will continue to do the dilutions each with a fresh pipette tip until I’m done. Then I will label 11 culture tubes of M. foliorum with the appropriate dilution and the 11th is the negative control. After I will infect the tubes with 10ul of the dilution and add 10 ul of phage buffer into the negative control. I will wait 10 minutes for infection. Lastly I will remove a tube of TA from the water bath and add CaCl2 and mix it with the infected bacteria. Then I will transfer the mixture on the labeled plate and gently spread the mixture on the top of the agar plate to let sit for 10 minutes to solidify. Then I will incubate the plates for at least 24 hours at 30-degrees
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