- Obtain the cylinder, cap, large T, small T, spout, and rubber hose, from the refrigerator. No, these are not the technical terms.
- Screw in the spout, attach the hose, and screw in the small T loosely.
- Rub vaseline along the edge of the insertion end of the large T, and insert into opening of the cylinder on the opposite side of the small T/spout , but leave open space for your resuspended cells.
- With cylinder upright and large T facing down, pour your resuspended cells into the open space. Pull the large T down until the top line of the resuspension is below the small hole that opens into the spout. Then screw the small T in tightly.
- Close the other side of the cylinder with the cap.
- With the small T screwed in loosely, gently tap the large T further into the cylinder until the resuspended cells just enter the spout and hose.
- Invert the apparatus such that the large T is on top, and position it in middle of the vise of the hydraulic press.
- Turn the large black-end handle to the right, make sure the max pressure (grey hand on the dial) is set at 20,000 psi, and press both "start" buttons to start closing the vise. Right before it's completely closed, reposition the cylinder such that it's exactly in the center. Then press both "start" buttons again to close the vise until the pressure has reached the maximum 20,000 psi.
- Get an Oak Ridge centrifuge tube or a 50ml tube, and hold it under the hose. Slowly loosen the small T. The lysed cells will begin to flow out of the hose into your tube; the flow rate should be no faster than individual drops or a very slow trickle.
- If the machine stops "kicking" or the pressure gauge drops, then you need to reapply pressure. First, tighten the small T again, and then press both "start" buttons until the pressure has reached the maximum 20,000 psi.
- Continue until all of the cell lysate has come out of the cylinder. Keep the end of the hose in the tube as you pull the black-end handle to the left to release the hydraulic press.
- Remove the cylinder, and then pour in your cell lysate, and repeat the process a second time.
to be finished