Phage DNA prep --- l phage

Grow l phage on plates

1. Grow host cells (e.g., Y1090, LE392) overnight in 5 ml of T-TYN/0.2% maltose/10 mM MgCl2 (or LB/0.2% maltose/10 mM MgSO4).

2. Dilute 2 ml of the overnight culture with 3 ml medium.

3. Infect by adding ~50,000 pfu of l phage.

4. Incubate at 37 C for 15-20 minutes.

5. Melt 10 ml T-TYN top agarose and place in 45-50 C water bath.

6. Mix the infected cells with T-TYN top agarose and pour onto 150 mm T-TYN agar plate.

7. Incubate at 37 C overnight.

8. Add 10 ml of SM to the plate. Incubate at 4 C for 2-3 hours on a Rock and Roll shaker.

9. Aspirate SM to a 50 ml tube.

10. Add 5 ml of SM to the plate. Incubate at 4 C for 30 minutes and aspirate SM to the 50 ml tube.

11. Add 5 ml chlorofom to the SM containing phages. Vortex.

12. Centrifuge and transfer the aqueous phase to a fresh tube.

Grow l phage in liquid culture

1a. Infect 2x 109 cells (2-3 ml mid-log phase host cells) with 107 pfu l phage.

2a. Incubate at 37 C for 15-20 minutes.

3a. Add infected cells to 50 ml NZCYM medium.

4a. Incubate at 37 C with shaking until all cells have lyzed.

5a. Add 2 ml chloroform and continue the shaking at 37 C for another 10-15 minutes.

Isolation of l phage

13. Add RNase A and DNase I to a final concentration of 1 mg/ml and incubate for 30 minutes at room temperature.

14. Add 2.9 g NaCl for each 50 ml of phage solution. Precipipate on ice for 30 minutes.

15. Centrifuge down bacteria debris for 10 minutes.

16. Transfer the supernatant to a clean tube and add PEG 8000 to the final concentration of 10%.

17. Precipitate phages on ice for 2 hours or 4 C overnight.

18. Spin down phage/PEG precipitates for 10 minutes.

19. Pour the supernatant and drain the remaining supernatnat by inverting the tube on a paper towel.

Purification of phage DNA

20. Resuspend the pellet in 10 ml SM and 10 ml chloroform.

21. Centrifuge to separate phases.

22. Transfer the supernatant to a fresh tube.

23. Extract twice with phenol/chloroform.

24. Precipitate phage DNA by adding 1 volume of isopropanol.

25. Pellet phage DNA by centrifugation.

26. Wash the pellet with 70% ethanol and air dry.

27. Resuspend the pellet in 100 ml TE with RNase A (20 mg/ml).