Preparation of phage particles from phage vectors

Preparation of phage particles from phage vectors

1. Pick up one phage vectors-containing colony with a sterile loop and put into 10 ml 2xTY + 10 µg/l tetracycline.
2. Shake at 200 rpm and 37 °C untill the OD₆₀₀~ 0.5.
3. Dilute bacterial culture into 200 ml 2xTY + 10 µg/l tetracycline (dilution 1:20).
4. Shake at 200 rpm and 30 °C approximately 20 hours.
5. Centrifuge the bacterial culture (10000 rpm, 30 min, 4 °C) and transfer the supernatant into 50 ml Falcon Tubes. Discard the pellet.
6. Add 1/5 volume PEG/NaCl (20% Polyethylene glycol 6000, 2.5 M NaCl) to the supernatant (50 ml PEG/NaCl to 200 ml supernatant). Mix well and leave 1 hour on ice.
7. Centrifuge (4000 rpm, 15 min, 4 °C) and discard the supernatant. White phage pellet should be at the bottom of the tube.
8. Resuspend phage pellet in 40 ml Millipore water and add 1/5 volume PEG/NaCl (10 ml). Mix well and leave 30 min on ice.
9. Repeat step 7.
10. Aspirate off any remaining drops of supernatant.
11. Resuspend the pellet in 1 ml 15% glycerol-containing PBS.
12. Centrifuge (13000 rpm, 2 min, room temperature) to remove most of the remaining cell debris.
13. Make aliquots of phage solution.
14. Freeze phage solution in liquid nitrogen and store at - 20 °C.

Preparation of phage particles from phagemids

1. Pick up one phagemid-containing colony with a sterile loop and put into 2 ml 2xTY + "100 µg/l ampicillin, 1 % glucose".
2. Shake at 200 rpm and 37 °C untill the OD₆₀₀ is 0.4 - 0.5.
3. Infect 1.5 ml bacterial culture with 1010 t.u/ml helper phage VCS M13 at 37 °C for 30 minutes.
4. Centrifuge (1300 rpm, 2 min, room temperature) and discard the supernatant
5. Resuspend bacterial pellet in 1 ml 2xTY + "100 µg/l ampicillin, 33 µg/ml" kanamicine and pour it into 50 ml 2xTY + "100 µg/l ampicillin, 33 µg/ml".
6. Shake at 200 rpm and 30 °C for 12-14 hours.
7. Centrifuge the bacterial culture (10000 rpm, 30 min, 4 °C) and transfer the supernatant into 50 ml Falcon Tubes. Discard the pellet.
8. Add 1/5 volume PEG/NaCl (20% Polyethylene glycol 6000, 2.5 M NaCl) to the supernatant (50 ml PEG/NaCl to 200 ml supernatant). Mix well and leave 1 hour on ice.
9. Centrifuge (4000 rpm, 15 min, 4 °C) and discard the supernatant. White phage pellet should be at the bottom of the tube.
10. Resuspend phage pellet in 40 ml Millipore water and add 1/5 volume PEG/NaCl (10 ml). Mix well and leave 30 min on ice.
11. Repeat step 7.
12. Aspirate off any remaining drops of supernatant.
13. Resuspend the pellet in 1 ml 15% glycerol-containing PBS.
14. Centrifuge (13000 rpm, 2 min, room temperature) to remove most of the remaining cell debris.
15. Make aliquots of phage solution.
16. Freeze phage solution in liquid nitrogen and store at - 20 °C.