LARGE SCALE PHAGE DNA PREP USING THE TL-100

LARGE SCALE PHAGE DNA PREP USING THE TL-100

For 500 ml Prep:

  1. Inoculate 100 ml of NZ (per 500 ml: 5.0 g NZ amine, 2.5 g NaCl, 0.5 g MgSO4 (anhydrous), 5 drops 10 M NaOH. 5 ml 10% yeast extract) in a 500 ml flask with an appropriate E. coli host (e.g. BNN45) for bacteriophage \ . Incubate overnight at 37 C with vigorous shaking.
  2. Read the OD600 of the culture. Calculate the cell concentration assuming that 1 OD600 = 8 x 108 cells/ml.
  3. Withdraw one aliquot of 1010 cells. Centrifuge at 4000g for 10 minutes at room temperature. Discard the supernatant.
  4. Resuspend the bacterial pellet in 3 ml of SM.
  5. Add 5 x 107 bacteriophage per 1010 bacterial cells.
  6. Incubate at 37 C for 20 minutes with intermittent shaking.
  7. Add \-infected aliquot of 1010 cells to 600 ml of NZ (pre-warmed to 37 C) in a 2 liter flask. Incubate at 37 C with vigorous shaking until lysis (about 9-12 hours).
  8. Add 10 ml of chloroform and continue shaking for additional 30 minutes.
  9. Chill the lysed culture to room temperature and add pancreatic DNase and RNase, both to a final concentration of 1 g/ml. Incubate for 30 minutes at room temperature.
  10. Add NaCl to a final concentration of 1 M (29.2 g/500 ml culture). Dissolve by swirling. Let stand on ice for 1 hour.
  11. Remove debris by centrifugation at 11,000g for 10 minutes at 4 C. Pool supernatant into a clean flask. [I have let the suspension set at 4 C overnight in the cold room and carefully poured off the supernatant, trying not to collect any of the settled cell debris.]
  12. Add solid polyethylene glycol (PEG 6000) to a final concentration of 10% w/v (i.e., 40 g/ 400 ml of supernatant). Dissolve by slow stirring.
  13. Cool in ice water and let stand for at least 1 hour (and not much more) to allow bacteriophage particles to precipitate.
  14. Recover the precipitated bacteriophage particles by centrifugation at 5000g for 45 minutes at 4 C [JA10 rotor]. Discard supernatant and stand centrifuge bottles in tilted position for 5 minutes to allow the remaining fluid to drain away from the pellet. Remove the fluid with a pipette.
  15. Gently resuspend pellet [usually coated on side of 500 ml centrifugation bottle] into minimal volume of TM, trying not to exceed 1 ml.
  16. Add equal volume of chloroform to bacteriophage suspension and vortex for 30 seconds. Seperate organic and aqueous phases by centrifugation at 1600g for 15 minutes at 4 C. Recover the aqueous phase.
  17. Add 1.0 ml of the aqueous phase carefully to the top of a glycerol step gradient in a 2.2 ml TLS55 polyallomer tube [before ice melts (sometimes ice from the second layer floats to the top, no real problem)]. To prepare the step gradient add 0.5 ml of 40% glycerol in TM, then freeze in dry ice-methanol bath. Once frozen, then layer 0.65 ml 5% glycerol in TM on top of the frozen bottom layer and freeze. Store at -20 C until use. Be certain that the tubes are balanced.
  18. Load tubes carefully into the TLS55 rotor. Spin at 50,000 rpm at 4 C for 1 hour [slow acceleration and slow deceleration].
  19. Decant aqueous portion of the tube [save just in case]. The bacteriophage should form a faint translucent pellet at the bottom of the tube. [After decanting the supernatant there is usually some residual TM-glycerol, that's OK].
  20. Prepare three CsCl solutions of 5.65 M (4.757 g / 5 ml), 4.85 M (4.080 g / 5 ml), and 3.26 M (2.744 g / 5 ml) in TM.
  21. Add about 0.2 ml of the 5.65 M CsCl solution to the bacteriophage pellet in the tube a mix gently [Because there is some residual TM-glycerol, don't let the volume go over 0.3 ml]. Then carefully layer 1.2 ml of 4.85 M CsCl solution. Finally, carefully layer 0.5 ml of the 3.26 M CsCl solution. Be certain that the tubes are balanced.
  22. Load the tubes into the TLS55 rotor and spin at 50,000 rpm at 20 C for 1 hour [slow acceleration, slow deceleration].
  23. Carefully unload tubes and locate the translucent band of bacteriophage which has floated up from the bottom. From the top of the tube remove with a pipette tip the excess CsCl-TM from the top to the band. Then collect the bacteriophage band and place in a seperate microfuge tube.
  24. Dialyze the collected bacteriophage fractions.
  25. Proceed with DNA extraction protocol.