Genetics - Quantitation of Bacteriophage P22
Jeff Lawrence (G02)
- Grow host strain overnight in LB.
- Dilute bacteriophage serially in 0.9% NaCl. To make
Add 100 µL bacteriophage to 900 µL 0.9% NaCl.
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.
Last Update: Thursday, 19-Jun-2014 11:54:42 PDT
This page has been viewed 167 times.
eckofoid at ucdavis.edu