|
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).
| 