User:Kalkao/notebook/Purifying T7 Particles

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This protocol was developed from the supplementary information of the "Refactoring bacteriophage T7" paper.

  1. Retrieve BL21 overnights.
  2. Place LB in the 37*C incubation room to warm up.
  3. Grow cells to a density of 1e8 to 1e9 cells/mL.
    • Put 40mL of LB into three armed flasks.
    • Put 2mL of culture into two of the flasks.
    • Place these two flasks in the 37*C shaking water bath.
    • Obtain two 2L flasks.
    • Pour 500mL of LB directly into each flask.
    • Be very careful and use sterile technique.
    • After about one and a half hours or when the 40mL cultures become fairly dense, put 40mL of culture in each flask from the armed flasks.
    • Place these flasks in the 37*C shaking water bath to incubate.
    • Check the OD of the flasks periodically.
  4. When the OD has reached between 0.3 and 0.4, add 5 drops of phage to each flask.
  5. Check the flasks every few minutes. When the flasks clear, add NaCl to the lysate to make the concentration 1 molar.
    • For the conditions specified above, add 29.22 grams of NaCl to each flask.
  6. Swirl the flasks to mix the salt in.
  7. Pour the cultures straight into large centrifuge tubes.
  8. Centrifuge these using the GS3 rotor for 10 minutes at 9000RPM.
    • Use water to balance the two tubes on a scale.
  9. Pour the supernatant into a large, wide 2800mL flask.
    • This can be stored overnight in the 4*C cold room, if needed.
  10. Add 10% w/v PEG to the supernatant.
    • For the conditions specified above, add 100 grams of PEG to the 2800mL flask (1L total volume).
  11. Swirl the flask to mix the PEG in.
    • It helps to do this in the 4*C cold room, so that the lysate stays cold.
    • Mix the PEG as thoroughly as possible- until the PEG is completely dissolved.
  12. Pour the lysate into a 2L flask.
  13. Place the flask in an ice bucket (with ice) for 1 hour.
    • It helps to put the ice bucket and flaks in the cold room for this hour.
  14. Obtain two plastic centrifuge test tubes and pour CsCl2 step gradients.
    • Pipette 1mL of p=1.43 CsCl2 into each tube.
    • Use 3mL syringes (w/ 20g 1/2 needles) to inject 1mL of p=1.53 CsCl2 into the bottom of each tube.
    • Use 3mL syringes again (w/ 20g 1/2 needles) to inject 1mL of p=1.62 CsCl2 into the bottom of each tube.
      • Be sure to use nitrile gloves because the CsCl2 is very concentrated and may harm your skin.
      • When injecting, begin slowly and gradually speed up.
      • Work slowly and carefully so the layers are not disrupted. Watch out for bubbles, which can also disturb the layers.
      • When handling needles, it is very important to avoid people and to avoid pointing the tip outwards in order to reduce the risk of poking someone.
  15. After the 1 hour incubation, pour the lysate into two 500mL centrifuge tubes.
  16. Centrifuge the tubes at 5000 RPM for 15 minutes in the GS3 rotor.
    • Water can be used again to balance the two tubes on a scale.
  17. When the centrifuge is done, pour the supernatant into the sink.
    • Take some extra time to touch the lip of the centrifuge tubes to a paper towel in order to remove as much PEG from the tube as possible.
  18. Resuspend the pellet of one tube in 7mL of T7 buffer.
    • Vortex this thoroughly.
  19. Use this suspension and to resuspend the pellet of the other tube.
    • Vortex thoroughly.
  20. Pour this new suspension into a 40mL centrifuge tube.
  21. Add 10mL of T7 buffer to the tube in order to dilute any leftover PEG.
  22. Balance the weight of this tube with another 40mL centrifuge tube.
  23. Centrifuge these tubes for 10 minutes at 5000 RPM, using the SA-600 rotor.
  24. After the spin, pour the supernatant into a cup.
    • It may also help to parafilm the cup and place it on ice.
  25. Use a 10mL syringe with a 20 g 1/2 needle to add phage to the top of the step gradient.
    • Slowly drip the supernatant down the side of each centrifuge test tube so that the layers are not disturbed.
    • Add 5mL of supernatant to each step gradient.
  26. Obtain the test tube holding apparatus that goes with the SW41T1-4 rotor.
  27. Use a syringe to slowly fill up each tube nearly to the lip with T7 buffer.
    • It may help to do this on a balance to make sure the tubes are the same weight.
    • Always check to make sure the weights are equal. Small differences in weight can damage the ultracentrifuge.
  28. Load the test tubes into two holders and place the lids on all the holders.
    • The lids can be screwed shut with a coin.
  29. Get the SW41T1-4 rotor and bring all of the apparatus to the ultracentrifuge room.
    • Be very careful when handling the rotor and apparatus because they are both very expensive.
  30. The rotor has two holes on the side of the botton piece that marks the location of two stubs that point out of the bottom of the rotor. These two stubs connect with two holes in the centrifuge. Use these markings to more smoothly align the rotor.
  31. Record the revolution number on the ultracentrifuge.
  32. Set the ultracentrifuge to speed: 30,000 RPM, time: 3 hours, and temperature: 4*C.
    • The time can be set for up to 5 hours.
  33. Set the acceleration and decceleration to "MAX."
  34. Hit the vacuum button.
    • The "750" sign will blink and then the "200" sign will turn on. The rotor will not spin faster than 3000RPM before the vacuum hits 200 microns.
  35. Hit the start button.
  36. Retrieve the centrifuge tubes when the spin is finished and handle the rotor and tube holders carefully.
  37. Put the rotor back in the cold room.
  38. Observe the phage band in the plastic centrifuge test tubes.
    • There should be 3 visible bands in the step gradient- the lowest one is the phage band (thin fine layer, whitish in color).
  39. Extract the phage from the test tube.
    • Use a 5mL syringe with a 26 g 3/8 needle.
    • Insert the needle horizontally through the plastic, just below the phage band.
    • Tilt the needle up into the phage band and extract slowly.
      • It is not necessary to move the needle around to extract phage.
      • Try not to extract more fluid than necessary.
      • Use nitrile gloves again during this step because when the needle is pulled out of the tube, the CsCl2 will drip out of the hole that was made.
  40. Eject the phage solution into a 1.7mL centrifuge tube and place it in the 4*C fridge.
  41. Clean the tube holding apparatus.
    • For each cap and each tube holder, do the following:
      • Spray twice with distilled water. Make sure to wash out the insides of the tube holders as well.
      • Spray twice with 70% ethanol. Be sure to wash out the insides of the tube holders as well.
      • Spray once with 95% ethanol. Be sure to wash out the inside of the tube holders as well.
      • When placing the tube holders back on the rack, be sure to put them back in an inverted position, to help the inside of the tube holders dry out.

This protocol is incomplete.

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