1. Prepare dry lipid mixture by lyophilization or evaporation.
2. Place the extruder stand / heating block onto a hot plate. Insert a thermometer into the well provided in the heating block. Switch the hot plate on, and allow to reach the desired temperature. Allow the temperature of the heating block to reach the desired value (approximately 15 minutes).
3. Hydrate lipid mixture using a suitable buffer for at at least 30 min. The lipid suspension should be kept above the phase transition temperature of the lipid during hydration and extrusion. To increase the efficiency of entrapment of water-soluble compounds, one may subject the hydrated lipid suspension to 3-5 freeze/thaw cycles by alternately placing the sample vial in a dry ice bath and warm water bath.
4. Once the sample is fully hydrated, load the sample into one of the gas-tight syringes and carefully place into one end of the mini-extruder.
   Note: To reduce the dead volume, pre-wet the extruder parts by passing a syringe full of buffer through the extruder; discard the buffer. New syringes may have tight fitting parts; to facilitate extrusion, pre-wet syringe barrel and plunger with buffer prior to inserting plunger into barrel.

Heating Block Update In our continuing effort to improve the design and functionality of the Avanti Mini-Extruder, we have detected a design flaw which can be corrected quickly by installation of the enclosed washer. On the black aluminum extruder heating block/stand, remove the hex screw holding the large clip which retains the extruder body. Place the stainless steel washer between the large clip base and the hex screw and install the hex screw in the heating block/stand. Tighten the hex screw (see photo). Once the stainless steel washer is in place, it will raise the height of the hex screw head and provide improved alignment of the extruder body with the syringe channels.

New Hamilton Syringes In our continuing effort to resolve workmanship quality issues with the syringe for the Avanti Mini-Extruder, Avanti has contracted The Hamilton Company to provide syringes for the extruder. The syringes provided by The Hamilton Company have a larger diameter glass barrel than previous syringes. Therefore it is necessary to loosen the syringe holding clips on the extruder heating block/stand to accommodate the larger syringes. Using a 2 mm hex wrench, slightly loosen the hex screws which secure the syringe holding clips until they will easily pass over the syringe barrel, making sure that sufficient pressure is applied to the barrel to provide complete contact with the heating block/stand for heat transfer. If the syringes do not lie flat in the heating block/stand syringe channel, rotate the syringe in the extruder clockwise until the syringe lies flat in the syringe channel. Important: never rotate the syringe in the extruder counterclockwise as this will loosen the needle assembly from the glass barrel causing sample leakage.

• Place the empty gas-tight syringe into the other end of the mini-extruder. Make sure the empty syringe plunger is set to zero; the syringe will fill automatically as the lipid is extruded through the membrane.
• Check the temperature of the heating block BEFORE placing the assembled extruder apparatus into the heating block. The temperature must be below 80°^C, or the syringes will be damaged.
• Insert the fully assembled extruder apparatus into the extruder stand.
  Insert the hex nut so that the apex of the hex nut points down.

Note: The extruder apparatus must be fully assembled before inserting in the heating block,
otherwise it will be damaged.

• Allow the temperature of the lipid suspension to equilibrate with the temperature of the heating block (approximately 5-10 minutes)
• Gently push the plunger of the filled syringe until the lipid solution is completely transferred to the alternate syringe.
  
  
• Gently push the plunger of the alternate syringe to transfer the solution back to the original syringe.
• Repeat previous two steps. A minimum of 4 times (total of 10 passes through membrane). In general, the more passes though the membrane, the more homogenous the lipid solution becomes.
  
  
• The final extrusion should fill the alternate syringe. This is to reduce the chances of contamination with larger particles or foreign material.
• After the final extrusion, remove the mini-extruder from the heating block.
• Remove the filled syringe from the extruder and inject the lipid solution into a clean sample vial. Important: When removing syringes, pull the syringe straight out of the extruder; removing at an angle could result in cracking the syringe.
• Store the vesicle preparation above the transition temperature of the lipid during the experiment. When not in use, store the vesicle solution at 4°^C. Do not freeze. Vesicle solutions are not stable in aqueous media for more than 3-4 days when stored at 4°^C. Storage of vesicle solutions at higher temperatures and pH <5 or >8 may reduce the lifetime of the vesicle suspension.
• Clean apparatus thouroully before using with a new lipid preparation.