Materials Required

• Chloroform
• Ethanol
• Dialysis buffer
• EDTA
• Equilibrated phenol
• SDS
• Phenol : chloroform (1:1)
• 3M Sodium acetate (pH 7.0)
• TE buffer
   

Procedure

1. Transfer the purified bacteriophage suspension into a dialysis tubing membrane which is sealed at one end with a clamp. Place the sealed tube in a beaker containing a 1000-fold volume of dialysis buffer.
2. Dialyze the bacteriophage suspension for 1 hour at room temperature with slow stirring.
3. Change the dialysis buffer and dialyze the bacteriophage suspension for an additional hour.
4. Carefully transfer the bacteriophage suspension into a centrifuge tube.
5. Add 0.5 M EDTA (pH 8.0) to a final concentration of 20 mM.
6.  Add proteinase K to a final concentration of 50 µg/ml.
7. Add SDS to a final concentration of 0.5%, and mix the solution by inverting the tube several times.
8.  Incubate the digestion mixture at 56°C for 1 hour and later cool it into room temperature.
9. Add an equal volume of equilibrated phenol to the digestion mixture, and then aqueous and organic phases are formed. Mix these phases by gently inverting the tube several times.
10.  Separate the phases by centrifugation at 3000g (5000 rpm in a Sorvall SS-34 rotor) for 5 minutes at room temperature. Use a wide-bore pipette to transfer the aqueous phase to a clean tube.
11.  Extract the aqueous phase once with a 1:1 mixture of equilibrated phenol and chloroform.
12.  Recover the aqueous phase as described above (Step 9), and repeat the extraction with an equal volume of chloroform. For large-scale preparations, proceed to Step 12. For smaller-scale quantities (bacteriophage from 50- to 100-ml cultures):
a. Recover the bacteriophage DNA by standard ethanol precipitation.
b. Store the solution for 30 minutes at room temperature.
c. Redissolve the DNA in an appropriate volume of TE (pH 7.6), and proceed to Step 14.
13. Transfer the aqueous phase to a dialysis sac.
14. Dialyze the preparation of bacteriophage DNA overnight at 4°C against three changes of a 1000-fold volume of TE (pH 8.0).
15.  Measure the absorbance of the solution at 260 nm and calculate the concentration of the DNA. 1 OD260 = 50 µg/ml of double-stranded DNA. A single particle of bacteriophage contains approx. 5 x 10-11 µg of DNA. The yield of bacteriophage DNA usually ranges from 500 µg to several mg per liter, depending on the titer of the bacteriophage in the lysed culture.
16. Check the integrity of the DNA by analyzing aliquots (0.5 µg) that are undigested or have been cleaved by appropriate restriction enzyme.  Analyze the DNAs by electrophoresis through a 0.7% agarose gel, using markers of an appropriate size.
17.  Store the stock of bacteriophage DNA at 4°C.