IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.3

Equipment

 * Glass vial 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
and or
 * 10ml of dodecane
 * 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
 * 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%

Preparing the lipid-oil suspension for the inner leaflet
 * 1) Place 25 µl of the 20 mg/ml DOPC or POPC solution in a glass vial. (Equipment should generally be made of glass and not plastic so as to prevent adsorption 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 debris into the lipid film)
 * 3) Put the vial in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
 * 4) Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml.
 * 5) Place the vial 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 dodecane.

Equipment

 * Small tubes
 * Aluminium foil
 * 20µl pipette + tips
 * 200µl pipette + tips
 * 1000µl pipette + tips
 * Tabletop centrifuge
 * Vortex machine
 * Syringe + stainless steel needle
 * Glass slide + cover slip
 * Optical microscope with phase contrast
 * Fluorescent microscope

Reagents

 * Commercial S30 E.coli extract
 * E.coli complete amino acid mixture
 * S30 premix without amino acid
 * S30 extract circular
 * Nuclease-free water
 * Plasmid DNA wtih pTet-GFP construct

Preparation of Reaction
 * 1) Add 5µl of E.coli complete amino acid mixture into a tube.
 * 2) Then add 20µl of S30 premix without amino acid.
 * 3) Next add 15µl of S30 extract circular.
 * 4) Add an appropriate volume of plasmid DNA depending on DNA concentration. (How many DNA molecules are needed in this step?)
 * 5) Finally add nuclease-free water to bring final volume to 100µl.

Formation of mono-layer vesicles
 * 1) Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension.
 * 2) Vortex gently for a few seconds. (This is to break up the small aqueous droplet to form an extract-oil emulsion)
 * 3) Leave to stand for a few minutes. (Microdroplets will be stabilized by a monolayer of phospholipids at the oil–extract interface)

Formation of bi-layer vesicles (Note: Alternatively, centrifuge at 30 x g for 20 min)
 * 1) Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids.
 * 2) Leave to stand for a few minutes. (A monolayer of phospholipids will form at the interface of the biphasic solution)
 * 3) Centrifuge at 120 x g for 10 min.

Collecting the vesicles: Ideally, the protocol should yield a few hundreds of vesicles and aggregates of 1 to a few tens of micrometers diameter. (Caution: Over-exposure of light under the microscope would bleach the GFP!!)
 * 1) Using a syringe with a stainless steel needle, collect some of the S30 premix.
 * 2) Expel some of the premix to remove all air from the syringe and needle. (Expelling most of the S30 premix would ensure a less diluted solution of vesicles)
 * 3) With the tip of the needle in the aqueous phase, gently expel the premix 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 solution 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 tube and store in the dark.
 * 8) Use optical microscopy to check that the vesicles obtained were not deformed or aggregated.