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 Celsius
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