HRV16 Purification

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PEG 8000 and NaCl for precipitation
0.25 M HEPES, 0.25 M NaCl, pH 7.5 stock buffer
0.5 M MgCl2
10 mg/ml DNAse in PBS (make fresh)
RNAse powder
Trypsin powder
0.1 M EDTA, pH 9.5
10% n-lauryl-Sarcosine
1 M NH4OH
30% sucrose in 20 mM Tris, 1 M NaCl, pH 7.4
10% potassium tartrate in 0.25 M HEPES, 0.25 M NaCl, pH 7.5
40% potassium tartrate in 0.25 M HEPES, 0.25 M NaCl, pH 7.5
0.1 M CaCl2, 0.3% PEG 8000 in 0.25 M HEPES, 0.25 M NaCl, pH 7.5

Harvest Infected Cells

  1. Collect infected cells and the media.
  2. Spin down at 3000 rpm, for 10 min at 4˚C (Rotanta 460 R)
  3. Resuspend pellet in 10 ml PBS.

Day 1 - Precipitate virus

  1. Lyse cells by freezing and thawing 3 times. Freeze bottles in dry ice with ethanol, thaw in 37˚C water bath.
  2. Homogenize the suspension.
  3. Spin at 10k, 10 min, 5˚C to remove cell debris (Sorvall SS-34 rotor)
  4. Determine the volume of the supernatant by weighing the supernatant in a capped pre-weighed sterile falcon tube (1 g ≈ 1 ml).
  5. In a beaker, add PEG 8000 to a final concentration of 5%. Add NaCl (MW = 58.44) to a final concentration of 0.5 M Stir overnight at 4˚C to precipitate the virus.

Day 2 - Purify and collect virus

  1. After stirring overnight, a cloudy white precipitant should be visible on the sides of the beaker.
    • Spin at 10K, 10 min, 5˚C to pellet virus (Sorvall SS-34 rotor).
    • Resuspend the precipitant in 10 ml of 0.25 M HEPES, 0.25 M NaCl, pH 7.5.
  2. Estimate the volume by weighing the suspension in a capped pre-weighed sterile falcon tube (1 g ≈ 1 ml).
  3. Add the following,
    • 5 µl of 1.0 M MgCl2 per ml of suspension to get 5 mM MgCl2.
    • 1 µl of 10 mg/ml DNAse stock per ml of suspension to get 10 µg/ml DNAse.
    • 50 µl of 10 mg/ml RNAse stock per ml of suspension to get 50 µg/ml RNAse.
    • (For 5 ml suspension, 25 µl MgCl2, 5 µl DNAse, 25 µl RNAse).
    • Leave at room temperature for 30 min.
  4. Add 0.8 mg Trypsin per ml of suspension. (40 mg for 5 ml suspension). Incubate at 35˚C for 10 min.
  5. After the incubating, the solution should become clear.
    • Add 150 µl of 0.1 M EDTA, pH 9.5 per ml of suspension to get 15 mM EDTA. (750 µl for 5 ml suspension)
  6. Estimate the new volume by weighing the suspension in the falcon tube.
  7. Add 100 µl of 10% n-lauryl-Sarcosine per ml of suspension to get 1% Sarcosine.
  8. Check the pH using a pH strip, pH should be ~8
    • If the color of the suspension turns yellow, add drops of 1 M NH4OH until the color returns to a red-orange color.
  9. Pour into 50 ml conical tubes and spin at 4,500 rpm, 10 min, 4˚C to remove some “junk.”
  10. Pour the supernatant into SW41 rotor tubes leaving about a centimeter of space at the top of the tubes.(each tube can take ~12ml)
    • Add 0.25 M HEPES, 0.25 M NaCl, pH 7.5 to the tubes if necessary so that only about 1 cm of space remains at the top.
  11. Add 30% sucrose in 20 mM Tris, 1 M NaCl, pH 7.4 to the bottom of the tube using a long glass pipette.
    • The dense sucrose will from a cushion below the supernatant.
    • Fill the tube until the liquid level reaches the neck of the tube. Balance the tube as well as the plug and cap. (The difference should be ≤0.05 g.)
  12. Spin in SW41 rotor at maximum speed, 41k, 2 hours, 4˚C.
  13. Suction off the supernatants. Brownish or grayish pellet near the bottom of each tube should be visible. Resuspend pellet in 0.5 ml of cold 0.25 M HEPES, 0.25 M NaCl, pH 7.5 using a long glass pipette to repeated blow the buffer over the pellets until they are dissolved. Pool the resuspended pellet.
  14. Optional: Spin 5k, 10 min, 5˚C to bring down bubbles from the resuspending of the pellets. Gently resuspend any residue on the side or bottom of this tube.
  15. Prepare 10% - 40% potassium tartrate gradient in rotor tubes. A linear gradient is prepared by using a gradient maker to mix the two stocks. (Use the gradient pump in Parrish lab.)
  16. Layer the resuspended pellets over the gradient by gently pipetting the suspension onto the top of the gradients. (No more than about 1-3 ml of suspension per gradient.).
    • Add small amounts of 0.25 M HEPES, 0.25 M NaCl, pH 7.5 to balance the tubes if necessary.
  17. Spin the SW41 rotor at 36k, 90 min, 4˚C.
  18. The virus should appear as a tight whitish or bluish band about half way down in the tube. It can be seen best by turning off the room lights and shining a small light down on the tube.
  19. Collect the band by puncturing the tube below the bland with a needle and syringe. Angle the needle up into the band and draw it out of the gradient into the syringe until it is no longer visible.
    • If more than one band is visible, the upper less dense band is probably empty virus particles. Do not collect this band. Do not mix it with intact virus particles from the lower more dense band.
    • If the band is not visible, puncture the bottom with the needle. Collect drops in a 24-well plate. (~50µl per drop).
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Adapted from Carol Bator Kelly's Prococol