Purification of Bacteriophages Using a Sucrose Density Gradient

JoVE Encyclopedia of Experiments

Mikrobiyoloji

0 görüntüleme • 3:36 dk • July 1st, 2026

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Begin with a suspension containing bacteriophages and host bacterial debris.

To purify the bacteriophages, sequentially add high-density and low-density sucrose solutions into an ultracentrifuge tube.

Using a gradient-forming instrument, mix the sucrose solutions by tilt-tube rotation to create a continuous density gradient.

Remove a small volume of sucrose gradient from the top, then carefully add the bacteriophage-debris mixture without disturbing the gradient.

Ultracentrifuge to facilitate differential migration of bacteriophages and bacterial debris based on their densities.

The bacteriophages localize as a thick, milky band halfway down the gradient.

Using a blunt cannula-assembled syringe, aspirate the phage band and transfer it to an ultracentrifuge tube.

Add buffer and mix gently.

Ultracentrifuge again to pellet the bacteriophages. Discard the supernatant.

Add buffer and incubate under cold conditions to allow resuspension of bacteriophages.

The purified bacteriophages are ready for further studies.

Prepare four 5 to 45 % sucrose gradients by pipetting 2.5 milliliters of 45 % sucrose into four ultracentrifuge tubes. Top the layer with approximately 2.6 milliliters of 5 % sucrose, stopping when the liquid reaches 2 millimeters from the rim of the tube.

Mix the gradients by tilt tube rotation using a gradient forming instrument for 43 seconds at 86 degrees and 23 RPM. Using a 1 milliliter pipette, add 500 microliters of the pooled phage suspension by bringing the tip into contact with the liquid surface and very slowly dispensing the volume onto the top of the sucrose gradient, being careful to not mix through rapid pipetting. Centrifuge at 70,000 times g for 20 minutes at 4 degrees Celsius.

Submerge a blunt cannula into the solution until the tip is centered on the very upper edge of the phage band. Remove the band by drawing on the syringe plunger until the majority of the phage band has been removed. Then add the sucrose volume with the suspended phage to three or four ultracentrifuge tubes.

Fill the tubes to between 3 and 4 millimeters from the top of the tube with cold 1x TM. Cover the tubes with paraffin film and invert to mix. Place the tubes back in an ultracentrifuge and spin at 145,000 times g for 1 hour at 4 degrees Celsius.

Quickly pour off the supernatant and drain upside down on disposable wipes. Divide 200 to 400 microliters of cold 1x TM across all pellets and resuspend overnight at four degrees Celsius. No shaking is required.

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