1. Add 1 ml of phage stock solution (109-1010 PFU/ml) to a 2 ml microcentrifuge tube.
2. Add 10 µl of DNase I (1 mg/ml) and 10 µl RNase A (5 mg/ml) to the tube.
3. Place the tube in a shaker thermoblock and incubate for 60 min at 37 °C.
4. Add 40 µl of 0.5 M EDTA-Na2 to the tube.
5. Add 2.5 µl of Proteinase K (20 mg/ml) to the tube.
6. Add 50 µl of 10 % SDS to the tube.
7. Place the tube in a shaker thermoblock and incubate for 1 h at 56 °C.
8. Let the tube cool down to room temperature (approx. 20 min).
9. Transfer the entire volume of the microcentrifuge tube into a 15 ml centrifuge tube.
10. Add 1.1 ml of phenol to the centrifuge tube, seal it with the lid and invert it 5 times.
11. Centrifuge the tube for 5 min at 22 °C and 4,000 g.
12. Transfer the upper aqueous phase into a new 15 ml centrifuge tube.
13. Add the same volume of phenol/chloroform in a ratio of 50/50 (e.g., for 0.9 ml of extracted volume in step 12, add 450 µl phenol and 450 µl chloroform).
14. Centrifuge the tube for 5 min at 22 °C and 4,000 g.
15. Transfer the upper aqueous phase into a new 15 ml centrifuge tube.
16. Add the same volume of chloroform (e.g., for 700 µl of extracted volume in step 15, add 700 µl chloroform).
17. Centrifuge the tube for 5 min at 22 °C and 4,000 g.
18. Repeat steps 16 and 17.
19. Transfer a maximum of 550 µl of the upper aqueous phase into a 2 ml microcentrifuge tube.
20. Add a maximum of 55 µl (1/10th of volume in step 19) sodium acetate to the tube.
21. Add a maximum of 1375 µl (2.5x volumes after step 20) ethanol (95 %).
22. Incubate the tube for 30 min on ice in a freezer (-20 °C).
23. Centrifuge the tube for 20 min at 4 °C and 14,000 g.
24. Remove the supernatant and add 2 ml of ethanol (70 %) to the tube.
25. Invert the tube 10 times and incubate it for 5 min at room temperature.
26. Centrifuge the tube for 5 min at 4 °C and 14,000 g.
27. Remove the supernatant and let the pellet dry on air (approx. 20 min).
28. Resuspend the pellet in 20 µl ddH2O.
29. Store the solution in a refrigerator at 4 °C until further use.
Using a spectrophotometer (e.g., NanoDrop 1000 Spectrophotometer; Thermo Fisher Scientific, Waltham, USA; cat. no.: ND-1000), the concentration of phage DNA and its purity can be evaluated.