Preparationofphageparticlesfromphagevectors

Pick up one phage vectors-containing colony with a sterile loop and put into 10 ml  2xTY + 10 µg/l tetracycline.

Shake at 200 rpm and 37 °C untill the OD₆₀₀~ 0.5.

Dilute bacterial culture into 200 ml 2xTY + 10 µg/l tetracycline (dilution 1:20).

Shake at 200 rpm and 30 °C approximately 20 hours.

Centrifuge the bacterial culture (10000 rpm, 30 min, 4 °C) and transfer the  supernatant into 50 ml Falcon Tubes. Discard the pellet.

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.

Centrifuge (4000 rpm, 15 min, 4 °C) and discard the supernatant. White phage  pellet should be at the bottom of the tube.

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.

Repeat step 7.

Aspirate off any remaining drops of supernatant.

Resuspend the pellet in 1 ml 15% glycerol-containing PBS.

Centrifuge (13000 rpm, 2 min, room temperature) to remove most of the remaining  cell debris.

Make aliquots of phage solution.

Freeze phage solution in liquid nitrogen and store at - 20 °C.

Pick up one phagemid-containing colony with a sterile loop and put into 2 ml  2xTY + "100 µg/l ampicillin, 1 % glucose".

Shake at 200 rpm and 37 °C untill the OD₆₀₀ is 0.4 - 0.5.

Infect 1.5 ml bacterial culture with 1010 t.u/ml helper phage VCS M13 at 37 °C for  30 minutes.

Centrifuge (1300 rpm, 2 min, room temperature) and discard the supernatant

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".

Shake at 200 rpm and 30 °C for 12-14 hours.

Centrifuge the bacterial culture (10000 rpm, 30 min, 4 °C) and transfer the  supernatant into 50 ml Falcon Tubes. Discard the pellet.

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.

Centrifuge (4000 rpm, 15 min, 4 °C) and discard the supernatant. White phage  pellet should be at the bottom of the tube.

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.

Repeat step 7.

Aspirate off any remaining drops of supernatant.

Resuspend the pellet in 1 ml 15% glycerol-containing PBS.

Centrifuge (13000 rpm, 2 min, room temperature) to remove most of the remaining  cell debris.

Make aliquots of phage solution.

Freeze phage solution in liquid nitrogen and store at - 20 °C.