BIO 351 PREPARATION AND PLAQUE ASSAY OF A PHAGE STOCK 2007 Özlem Yalçın Sunay Usluer Sunay Usluer M. Aslı Kayserili Taliha Paşaoğlu

QUIZ: 1. What is the purpose of CaCl2 addition into our phage titer mixture? 2. If you count 100 plaques in your plate, what is the pfu/ul of your stock? (hint: you prepared your dilutions in a total volume of 200ul and put 100ul into your phage titer mixture) (hint: you prepared your dilutions in a total volume of 200ul and put 100ul into your phage titer mixture)

Bacteriophage: Viruses that attack bacteria Viruses that attack bacteria Two types of life cycle: Two types of life cycle: 1.Lytic cycle:  The process beginning with a single phage genome and release of new phage progeny 2. Lysogenic cycle:  DNA is integrated into the host chromosome  The inserted phage genome or prophage is passively replicated as part of the bacterial chromosome

Lytic cycle

Lysogenic cycle:

Today’s Experiment: I. Preparation of Phage lysate On each bench, there are 2 plates which contains phage infected bacteria culture. On each bench, there are 2 plates which contains phage infected bacteria culture. Add 1 ml LB medium on each plate Add 1 ml LB medium on each plate By using a glass spreader, break up the soft agar By using a glass spreader, break up the soft agar Scrap the soft agar into cups Scrap the soft agar into cups By using a loop, pour the lysate into 15ml falcon By using a loop, pour the lysate into 15ml falcon Centrifuge falcons at 5000rpm for 15min. at 4 0 C Centrifuge falcons at 5000rpm for 15min. at 4 0 C By using the syringe, take the supernatant (liquid part) By using the syringe, take the supernatant (liquid part) Take off the needle part, Take off the needle part, Attach plunger to the filter WITHOUT TOUCHING THE TIP OF THE FILTER !!!! Attach plunger to the filter WITHOUT TOUCHING THE TIP OF THE FILTER !!!! Filter the lysate into 1.5ml eppendorf. Label it with group name and date Filter the lysate into 1.5ml eppendorf. Label it with group name and date

Today’s Experiment: II. Phage titer of your phage stock Label four 1.5ml eppendorf for 10 -2, 10 -4, 10 -6, Label four 1.5ml eppendorf for 10 -2, 10 -4, 10 -6, Prepare serial dilution of your phage stock, in a total of 200ul Prepare serial dilution of your phage stock, in a total of 200ul Label four 15ml falcon tube for 10 -2, 10 -4, 10 -6, Label four 15ml falcon tube for 10 -2, 10 -4, 10 -6, In each 15ml falcon, put 250ul of your bacteria (sensitive strain, E.Coli) In each 15ml falcon, put 250ul of your bacteria (sensitive strain, E.Coli) Add X ul CaCI2 (5mM) into each 15ml falcon Add X ul CaCI2 (5mM) into each 15ml falcon  phage to adsorb to E.Coli Add 100ul of your serial dilutions into your 15ml falcon tubes Add 100ul of your serial dilutions into your 15ml falcon tubes Add 3ml soft agar Add 3ml soft agar Vortex Vortex Immediately pour onto LB agar plates Immediately pour onto LB agar plates Incubate at 37 0 C overnight Incubate at 37 0 C overnight Count the plaques. Calculate the pfu/ul for your stock!!! Count the plaques. Calculate the pfu/ul for your stock!!!

What is a plaque? Each small clear area  plaque