IGEM:IMPERIAL/2007/Wet Lab/Protocols/VF1.1

Equipment

 * Nitrogen tap + plastic tubing
 * Desiccator connected to a vacuum
 * 100ml glass bottle
 * Sonicator with medium-sized probe
 * Ice bath
 * 25°C incubator
 * Pipette + pipette tips (1000µl)

Reagents

 * 10ml dodecane
 * 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%

Procedure
Preparing the lipid-oil suspension for the inner leaflet
 * 1) Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
 * 2) With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
 * 3) Put the bottle in a desiccator connected to a vacuum for 1h.
 * 4) Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
 * 5) Set the sonicator probe to pulse 1, timer at 30mins.
 * 6) Put the bottle containing the suspension in the ice bath.
 * 7) Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
 * 8) Sonicate the suspension for 30mins.
 * 9) Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil.

Equipment

 * Magnetic stirrer
 * Centrifuge + 1-inch glass centrifuge tubes
 * Pipette + pipette tips (200µl, 1000µl)
 * 50ml glass tube
 * 5ml syringe
 * Long 16-gauge stainless steel needle

Reagents

 * 10ml ddH2O
 * Tris buffer
 * NaCl
 * Reporter

Procedure
Emulsifying the aqueous solution (while the interface settles)
 * 1) Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
 * 2) Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
 * 3) Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A.
 * 4) Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
 * 5) Gently stir the mixture with a magnetic stir bar for 3h.

Preparing the interface (to be done while the emulsion is mixing)
 * 1) Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube.
 * 2) Leave for 2–3h for lipids to achieve the coverage of the interface surface.

Forming the vesicles
 * 1) Pour 100µl of the inverted emulsion over the interface.
 * 2) Centrifuge at 120g for 10min.

Collecting the vesicles
 * 1) Using a 5ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
 * 2) Expel some of the solution to remove all air from the syringe and needle.
 * 3) With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe.
 * 4) Gently recirculate the buffer several times.
 * 5) Aspirate most of the solution into the syringe, and remove the needle from the solution.
 * 6) Wipe the tip of the needle clean.
 * 7) Unload the vesicle suspension into its final container.

(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)