Genetics - Quantitation of Bacteriophage P1


Jeff Lawrence (G11)

1. Grow host cells overnight at 37°C in LB.

2. Add 500 µL cells to 5 mL LB. Incubate at 37°C for 60 min with shaking.

3. Centrifuge cells for 8 min at 6000 rpm at 4°. Discard supernatant.

4. Resuspend cells in 2.5 mL (1/2 original volume) of:
	5 mM	CaCl2	500 µL	1 M CaCl2
	10 mM	MgSO4	1 µL l	1 M MgSO4
			99 mL	H2O
5. Serially dilute P1-vir bacteriophage in:
	10 mM	Tris, pH 7.5	500 µL	1 M Tris, pH 8.0
	10 mM	MgSO4		500 µL	1 M Tris, pH 7.0
				1 mL	1 M MgSO4
				98 mL	H2O
6. Mix 100 µL cells and 100 µL bacteriophage dilution. Incubate at 30°C for 30 min.

7. Mix with 2.5 mL P1 Top Agar. Make as follows:
	1X	LB top agar	10 g	Tryptone
	5 mM	CaCl2		5 g	Yeast Extract
	10 mM	MgSO4		5 g	NaCl
				7 g	Agar
				5 mL	CaCl2
				10 mL	MgSO4
				985 mL	H2O

	a) Siphon solution into 100 mL autoclaved graduated cylinder 
	and dispense into 10 bottles while stirring.

	b) Autoclave for 20 min.
8. Vortex tube and plate on LB plate warmed to room temperature.

9. Incubate at 37°C overnight.

12. Count plaques. Multiply the number of plaques by 10(1+Dilution) to calculate the titer of the original bacteriophage stock. For example:
 	200 plaques on the 10-7 dilution 
	yields (2 x 102 pfu/100 µL)*(10)*(107) = 2 x 1010 pfu/mL
13. Calculate titer of transducing particles as 1% the titer of pfu's for strain P1-vir.
Last Update: Thursday, 19-Jun-2014 11:54:44 PDT
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Eric Kofoid eckofoid at ucdavis.edu