Genetics - Quantitation of Bacteriophage P22

Jeff Lawrence (G02)

  1. Grow host strain overnight in LB.

  2. Dilute bacteriophage serially in 0.9% NaCl. To make dilution:
  3. 	Add 100  µL bacteriophage to 900  µL 0.9% NaCl.
    	Repeat 7 times, making a stock at 10-8 original concentration.
  4. Melt λ top agar in microwave and keep at 45 - 55°C.

  5. Add 100 µL cells to 100 µL phage dilution.

  6. Add 2.5 mL melted λ top agar.

  7. Quickly vortex the tube and flame the edge.

  8. 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.

  9. Rotate the plate to evenly distribute the agar. Cover the plate.

  10. Allow the plate to stand for 2 - 5 min to allow agar to harden.

  11. Invert plate and place at 37°C overnight.

  12. Count bacteriophage. Multiply the number of plaques by 10(1+Dilution) to calculate the titer of the original bacteriophage stock. For example:
  13. 	200 plaques of the 10-8 dilution yields 
    	(2 x 102 pfu/0.1 mL)*(10)*(108) = 2 x 1011 pfu/mL
  14. Calculate titer of transducing particles as 1/3 the titer of pfu's for strain P22 int-205 HT-101.

Last Update: Thursday June 19 2014
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