Genetics - Quantitation of Bacteriophage P22
Jeff Lawrence (G02)
Grow host strain overnight in LB.
Dilute bacteriophage serially in 0.9% NaCl. To make
dilution:
Add 100 µL bacteriophage to 900 µL 0.9% NaCl.
Vortex.
Repeat 7 times, making a stock at 10-8 original concentration.
Melt λ top agar in microwave and keep at 45 - 55°C.
Add 100 µL cells to 100 µL phage dilution.
Add 2.5 mL melted λ top agar.
Quickly vortex the tube and flame the edge.
Dump tube contents onto λ plates prewarmed to room temperature. Do not
warm plates to 37°C or the agar will take forever to harden.
Rotate the plate to evenly distribute the agar. Cover the plate.
Allow the plate to stand for 2 - 5 min to allow agar to harden.
Invert plate and place at 37°C overnight.
Count bacteriophage. Multiply the number of plaques by
10(1+Dilution)
to calculate the titer of the original bacteriophage stock. For example:
200 plaques of the 10-8 dilution yields
(2 x 102 pfu/0.1 mL)*(10)*(108) = 2 x 1011 pfu/mL
Calculate titer of transducing particles as 1/3 the titer of pfu's for
strain P22 int-205 HT-101.