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Biomacromolecules
 
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Phage particles

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.
 

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© 2014 ETH Zurich | Imprint | Disclaimer | 13 December 2005
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