DNA Preparation -Bacteriophage Lambda Particles

Jeff Lawrence

1. Isolate 1 large, fresh plaque and add to 100 mL fresh overnight E. coli C600. Incubate at 37° for 20 min. Add 5 mL NZ1:

	10 g	NZ Amine (Casein hydrolysate)
	5 g	NaCl
	0.25 g	MgSO4*7H₂O
	1 L	H₂O

2. Incubate at 37° until lysis occurs, 3-6 hr. Add 50 ng DNase (50 µL of a fresh 1:1000 dilution of a 1 mg/ml stock). Incubate at 37° for 30 min with shaking.

3. Centrifuge at 2.5 krpm (1300 g) for 30 min in tabletop centrifuge.

4. Store 50 mL of lysate over 2 mL Chloroform as permanent stock.

5. Add 4 mL 50/50 DE-52 slurry to a sterile column and allow to settle. Rinse column with 5 mL TE and allow effluent to pass.

6. Add remaining lysate to column and allow effluent to pass. Add 5 mL Chase buffer:

	10 mM	Tris. pH 8.0	(1 mL	1 M Tris, pH 8.0)
	10 mM	MgOAc₂		(1 mL	1 M MgOAc₂)
	60 mM	EDTA, pH 8.0	(12 mL	500 mM EDTA, pH 8.0)
				(86 mL	ddH₂O)

7. After effluent passes, add 1 mL Elution buffer:

	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	50 mM	MgOAc₂		(5 mL	1 M MgOAc₂)
				(94 mL	ddH₂O)

8. After effluent passes, place columns over microcentrifuge tubes. Add 6 mL Elution buffer and collect the column effulent.

9. Store concentrated phage at 4° in the dark. Stock may be used to prepare DNA.

Last Update: Thursday, 12-Dec-2024 00:55:19 UTC

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Eric Kofoid eckofoid at ucdavis.edu