IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.3: Difference between revisions
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====Reagents==== | ====Reagents==== | ||
* 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0% | * 10ml of dodecane | ||
and | |||
* 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0% | |||
or | |||
* 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 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''' | '''Preparing the lipid-oil suspension for the inner leaflet''' | ||
# 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 | # 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)''' | ||
# 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)''' | # 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)''' | ||
# Put the vial in a desiccator connected to a vacuum for 1hr. '''(This is to remove the chloroform)''' | # Put the vial in a desiccator connected to a vacuum for 1hr. '''(This is to remove the chloroform)''' | ||
# Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml | # Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml. | ||
# Place the vial containing the suspension in the ice bath | # Place the vial containing the suspension in the ice bath. | ||
# Sonicate suspension for 30 min (Pulse 1, ~10 Amp). '''(This is to disperse the phospholipids)''' | # Sonicate suspension for 30 min (Pulse 1, ~10 Amp). '''(This is to disperse the phospholipids)''' | ||
# Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in dodecane | # Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in dodecane. | ||
===Day 2=== | ===Day 2=== | ||
====Equipment==== | ====Equipment==== | ||
* | * Small tubes | ||
* Aluminium foil | * Aluminium foil | ||
* 20µl pipette + tips | |||
* 200µl pipette + tips | * 200µl pipette + tips | ||
* 1000µl 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==== | ====Reagents==== | ||
* Commercial S30 E.coli extract | * Commercial S30 E.coli extract | ||
** E.coli complete amino acid mixture | ** E.coli complete amino acid mixture | ||
* | ** S30 premix without amino acid | ||
* Plasmid DNA | ** S30 extract circular | ||
** Nuclease-free water | |||
* Plasmid DNA wtih pTet-GFP construct | |||
''' | '''Preparation of Reaction''' | ||
# | # Add 5µl of E.coli complete amino acid mixture into a tube. | ||
# | # Then add 20µl of S30 premix without amino acid. | ||
# | # Next add 15µl of S30 extract circular. | ||
# | # Add an appropriate volume of plasmid DNA depending on DNA concentration. '''(How many DNA molecules are needed in this step?)''' | ||
# | # Finally add nuclease-free water to bring final volume to 100µl. | ||
''' | '''Formation of mono-layer vesicles''' | ||
# | # Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension. | ||
# | # Vortex gently for a few seconds. '''(This is to break up the small aqueous droplet to form an extract-oil emulsion)''' | ||
''( | # 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''' | '''Formation of bi-layer vesicles''' | ||
# | # Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids. | ||
# Centrifuge at 120 x g for 10 min | # Leave to stand for a few minutes. '''(A monolayer of phospholipids will form at the interface of the biphasic solution)''' | ||
# Centrifuge at 120 x g for 10 min. | |||
''(Note: Alternatively, centrifuge at 30 x g for 20 min)'' | ''(Note: Alternatively, centrifuge at 30 x g for 20 min)'' | ||
'''Collecting the vesicles:''' | '''Collecting the vesicles:''' | ||
# Using a | # Using a syringe with a stainless steel needle, collect some of the S30 premix. | ||
# Expel some of the | # 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)''' | ||
# With the tip of the needle in the aqueous phase, gently expel the | # 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)''' | ||
# Gently recirculate the | # Gently recirculate the solution several times. | ||
# Aspirate most of the solution into the syringe, and remove the needle from the solution. '''(Be careful not to aspirate the lipid-oil suspension)''' | # Aspirate most of the solution into the syringe, and remove the needle from the solution. '''(Be careful not to aspirate the lipid-oil suspension)''' | ||
# Wipe the tip of the needle clean. | # Wipe the tip of the needle clean. | ||
# Unload the vesicle suspension into a | # Unload the vesicle suspension into a tube and store in the dark. | ||
# Use optical microscopy to check that the vesicles obtained were not deformed or aggregated. Ideally, the protocol should yield | # Use optical microscopy to check that the vesicles obtained were not deformed or aggregated. | ||
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!!)''' | |||
===Notes=== | ===Notes=== | ||
* Time required for Day 1: ~ 2h; for Day 2: ~ | * Time required for Day 1: ~ 2h; for Day 2: ~2h. | ||
* The protocol is based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid= | * The protocol is based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15591347 A vesicle bioreactor as a step toward an artificial cell assembly] by Vincent Noireaux and Albert Libchaber. | ||
*Modifications to protocol: | *Modifications to protocol: | ||
** The original protocol uses | ** The original protocol uses mineral oil instead of dodecane | ||
** The original protocol uses | ** The original protocol uses egg lecithin instead of DOPC or POPC | ||
** The original protocol | ** The original protocol does not use Span 80 | ||
Latest revision as of 04:59, 3 September 2007
1.3 Protocol for Adding Cell Extract into Vesicles
Day 1
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
- 10ml of dodecane
and
- 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
or
- 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%
Preparing the lipid-oil suspension for the inner leaflet
- 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)
- 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)
- Put the vial in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
- Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml.
- Place the vial containing the suspension in the ice bath.
- Sonicate suspension for 30 min (Pulse 1, ~10 Amp). (This is to disperse the phospholipids)
- Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in dodecane.
Day 2
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
- Add 5µl of E.coli complete amino acid mixture into a tube.
- Then add 20µl of S30 premix without amino acid.
- Next add 15µl of S30 extract circular.
- Add an appropriate volume of plasmid DNA depending on DNA concentration. (How many DNA molecules are needed in this step?)
- Finally add nuclease-free water to bring final volume to 100µl.
Formation of mono-layer vesicles
- Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension.
- Vortex gently for a few seconds. (This is to break up the small aqueous droplet to form an extract-oil emulsion)
- 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
- Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids.
- Leave to stand for a few minutes. (A monolayer of phospholipids will form at the interface of the biphasic solution)
- Centrifuge at 120 x g for 10 min.
(Note: Alternatively, centrifuge at 30 x g for 20 min)
Collecting the vesicles:
- Using a syringe with a stainless steel needle, collect some of the S30 premix.
- 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)
- 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)
- Gently recirculate the solution several times.
- Aspirate most of the solution into the syringe, and remove the needle from the solution. (Be careful not to aspirate the lipid-oil suspension)
- Wipe the tip of the needle clean.
- Unload the vesicle suspension into a tube and store in the dark.
- Use optical microscopy to check that the vesicles obtained were not deformed or aggregated.
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!!)
Notes
- Time required for Day 1: ~ 2h; for Day 2: ~2h.
- The protocol is based on A vesicle bioreactor as a step toward an artificial cell assembly by Vincent Noireaux and Albert Libchaber.
- Modifications to protocol:
- The original protocol uses mineral oil instead of dodecane
- The original protocol uses egg lecithin instead of DOPC or POPC
- The original protocol does not use Span 80