IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.2

Equipment

 * 100ml glass beaker x1
 * Gilsson pipette (200µl) + pipette tips
 * Nitrogen tap
 * 1000µl pipette tip
 * Desiccator connected to a vacuum
 * 25ml Glass pipette x1
 * Sonicator with medium-sized probe
 * 25°C incubator

Reagents
(Note: Alternatively, use POPC and dodecane)
 * 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
 * Mineral oil

Preparing the lipid-oil suspension for the inner leaflet
 * 1) Place 125 µl of the 20 mg/ml DOPC solution in a 100-ml glass beaker. (Equipment should generally be made of glass and not plastic so as to prevent adhesion of lipid molecules to plastic surface)
 * 2) Using plastic tubing and a 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film. (Rubber tubes are not recommended as they are more likely to emit debis into the lipid film)
 * 3) Put the beaker in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
 * 4) Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
 * 5) Place the beaker containing the suspension in the ice bath
 * 6) Sonicate suspension for 30 min (Pulse 1, ~10 Amp). (This is to disperse the phospholipids)
 * 7) Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil

Equipment

 * Magnetic stirrer
 * Aluminium foil
 * 200µl pipette + tips
 * 1000µl pipette + tips
 * 50ml 1-inch diameter glass centrifuge tube
 * 1-inch diameter tabletop centrifuge
 * 5ml syringe + long 16-gauge stainless steel needle x1
 * Test tube x1

Reagents

 * Solution A
 * Tris buffer
 * NaCl
 * ddH20
 * GFP standard Solution

Preparation of Solution A
 * 1) Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4

Emulsifying the Aqueous Solution
 * 1) Separate about 5 ml of the lipid-oil suspension into a glass container. (For the interface preparation)
 * 2) Prepare 10X GFP standard solution with Solution A to form 1ml aqeous solution.
 * 3) Add 250 µl of the aqeous solution to the 45ml lipid-oil suspension in mineral oil.
 * 4) Wrap the beaker in aluminium foil. (This is to prevent bleaching of GFP)
 * 5) Gently stir the mixture with a magnetic stir bar for 3 hours.

Preparing the interface (While emulsion is mixed) (Note: This step can been modified to use 2ml of emulsified solution instead of the lipid-oil suspension)
 * 1) Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.
 * 2) Leave for 2–3 h for lipids to achieve the coverage of the interface surface (>3h and the lipid may start to clump together)

Formation of bi-layer vesicles (Note: Alternatively, centrifuge at 30 x g for 20 min)
 * 1) Pour 100 µl of the inverted emulsion over the interface. (Note: This step is omitted if 2ml of emulsified solution is used instead of the lipid-oil suspension)
 * 2) Centrifuge at 120 x g for 10 min

Collecting the vesicles:
 * 1) Using a 5-ml 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. (Expelling most of the Solution A would ensure a less diluted solution of vesicles)
 * 3) With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe. (This prevents the extraction of the lipid-oil suspension when the needle is plunged into the tube)
 * 4) Gently recirculate the buffer several times.
 * 5) Aspirate most of the solution into the syringe, and remove the needle from the solution. (Be careful not to aspirate the lipid-oil suspension)
 * 6) Wipe the tip of the needle clean.
 * 7) Unload the vesicle suspension into a test tube and store in the dark.
 * 8) Use optical microscopy to check that the vesicles obtained were not deformed or aggregated. Ideally, the protocol should yield ~109 vesicles of 1µm diameter. (Caution: Over-exposure of light under the microscope would bleach the GFP!!)