BIO 351BIO 351PREPARATION AND PLAQUE PREPARATION AND PLAQUE

ASSAY OF A PHAGE STOCKASSAY OF A PHAGE STOCK

20072007Özlem YalçınÖzlem Yalçın

Sunay UsluerSunay UsluerM. Aslı KayseriliM. Aslı KayseriliTaliha PaşaoğluTaliha Paşaoğlu

QUIZ:QUIZ:1.1. What is the purpose of CaCl2 addition What is the purpose of CaCl2 addition

into our phage titer mixture?into our phage titer mixture?

2.2. If you count 100 plaques in your 10If you count 100 plaques in your 10-4-4 plate, what is the pfu/ul of your stock?plate, what is the pfu/ul of your stock?

(hint: you prepared your dilutions in a (hint: you prepared your dilutions in a total volume of 200ul and put 100ul total volume of 200ul and put 100ul into your phage titer mixture)into your phage titer mixture)

Bacteriophage: Bacteriophage:

• Viruses that attack bacteriaViruses that attack bacteria

• Two types of life cycle:Two types of life cycle:

1.Lytic cycle:1.Lytic cycle:

The process beginning with a single The process beginning with a single phage genome and release of new phage genome and release of new phage progenyphage progeny

2. Lysogenic cycle:2. Lysogenic cycle:

DNA is integrated into the host DNA is integrated into the host chromosomechromosome

The inserted phage genome or prophage The inserted phage genome or prophage is passively replicated as part of the is passively replicated as part of the bacterial chromosomebacterial chromosome

Lytic cycleLytic cycle

www.bact.wisc.edu/themicrobialworld/Virus5c.jpgwww.bact.wisc.edu/themicrobialworld/Virus5c.jpg

Lysogenic cycle:Lysogenic cycle:

www.bact.wisc.edu/themicrobialworld/Viruses6.jpg

Today’s Experiment:Today’s Experiment:

I. Preparation of Phage lysateI. Preparation of Phage lysate

• On each bench, there are 2 plates which contains phage On each bench, there are 2 plates which contains phage infected bacteria culture.infected bacteria culture.

• Add 1 ml LB medium on each plateAdd 1 ml LB medium on each plate• By using a glass spreader, break up the soft agarBy 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 falconBy using a loop, pour the lysate into 15ml falcon• Centrifuge falcons at 5000rpm for 15min. at 4Centrifuge falcons at 5000rpm for 15min. at 400CC• 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 Attach plunger to the filter WITHOUT TOUCHING THE TIP WITHOUT TOUCHING THE TIP

OF THE FILTEROF THE FILTER !!!! !!!!• Filter the lysate into 1.5ml eppendorf. Label it with group Filter the lysate into 1.5ml eppendorf. Label it with group

name and datename and date

Today’s Experiment:Today’s Experiment:

II. Phage titer of your phage stockII. Phage titer of your phage stock• Label four 1.5ml eppendorf for 10Label four 1.5ml eppendorf for 10-2-2, 10, 10-4-4, 10, 10-6-6, 10, 10-8-8 • Prepare serial dilution of your phage stock, in a total of 200ulPrepare serial dilution of your phage stock, in a total of 200ul• Label four 15ml falcon tube for 10Label four 15ml falcon tube for 10-2-2, 10, 10-4-4, 10, 10-6-6, 10, 10-8-8 • In each 15ml falcon, put 250ul of your bacteria (sensitive In each 15ml falcon, put 250ul of your bacteria (sensitive

strain, E.Coli) strain, E.Coli) • Add Add XX ul CaCI2 (5mM) into each 15ml falcon ul CaCI2 (5mM) into each 15ml falcon phage to adsorb to E.Coli phage to adsorb to E.Coli • Add 100ul of your serial dilutions into your 15ml falcon tubesAdd 100ul of your serial dilutions into your 15ml falcon tubes• Add 3ml soft agarAdd 3ml soft agar• VortexVortex• Immediately pour onto LB agar platesImmediately pour onto LB agar plates• Incubate at 37Incubate at 3700C overnightC 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?What is a plaque?

Each small clear area plaque