Genetics - Amplification of Bacteriophage λ


Jeff Lawrence (G21)

1. Grow E. coli C600 overnight at 37° in 5 mL of liquid LB+ 0.1% maltose. The maltose induces the lamB receptor gene. LB is:
	10 g 	Tryptone
	5 g 	Yeast Extract
	5 g 	NaCl
	1 L 	Water
2. With the small end of a sterile Pasteur pipette, pick a single plaque and deposit it in a sterile tube.

3. Add 100 mL fresh overnight cells. Incubate at 37° for 30 min to adsorb phage to cells..

4. Add 5 mL of LB+10 mM MgSO4. Make as : (yeast extract is optional).
	10 g 	Tryptone
	5 g 	Yeast Extract
	5 g 	NaCl
	1 L 	Water
	Autoclave until sterile in 100 mL bottles.  

	To each 100 mL aliquot, add:
	1 mL	1M MgSO4
5. Incubate at 37° with vigorous shaking for 4-6 hours or until lysis occurs. Lysate will be clear. If the culture clears too quickly (1-2 hours), too much phage was added. If it does not clear, add more phage.

6. Spin at 8 Krpm for 10 min to pellet cell debris and store over 50 µL chloroform.



Last Update: Thursday, 19-Jun-2014 11:54:45 PDT
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Eric Kofoid eckofoid at ucdavis.edu