- Centrifuge the semen, buffer and cushion at 1750 – 2000 RPM for 20 minutes (approx. 1000 x g). Make sure that the tubes are "balanced" so that there is equal weight across the rotor. You can use a tube filled with the same volume of water if you have an unequal number of tubes.
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- Remove cushion solution if still visible as a clear layer at the bottom. The cushion solution is removed with a pipet. Remove the supernatant. The supernatant is usually removed with an aspirator. Carefully transfer the sperm pellet to a small tube.
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Removing cushion with Pasteur pipette |
Water aspirator uses water flow to form a vacuum. This device is used to remove large volumes like the supernatant. Care must be taken not to aspirate the sperm pellet.
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Sperm pellet after cushion buffer and supernatant are removed. The sperm pellet is at the bottom of the tube and there is some centrifugation buffer remaining on top of the pellet. |
