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- Protocol by Vincent Noireaux, University of Minnesota, School of Physics and Astronomy
Phospholipid preparation: eggPC (Avanti 840051P, MW = 770)
- Note: Special order phospholipids in small aliquots (~25 mg), as the lipids in the bottle do not last long after opening.
- 10mg + 1ml mineral oil, vortex strongly, heat at 50C for 10 minutes.
- Prepare a sub stock at 2mg/ml (200μl at 10mg/ml + 800μl mineral oil).
- Heat at 50C for 1 hour.
- Incubate at RT for 6 hours.
Reaction composition for 90μl (100% volume):
|2.7μl||Mg-glutamate at 100mM||(3mM final here) depends on batch|
|1.8μl||K-glutamate at 3M||(60mM final here) depends on batch|
|22.5μl||AA at 6mM|
|2μl||BSA-RITC at 10mg/ml||(3.33μM final) for visualization|
|0.9μl||pBEST-OR2-OR1-Pr-UTR1-sigma28 at 20nM||(0.2nM final)|
|9μl||pBEST-Ptar-UTR1-AH-eGFP at 50nM||(5nM final)|
|4.5μl||PEG at 40%||(2% final)|
Feeding solution 1 for 180μl (no PEG + extract): PEG interferes with vesicle formation
|20μl||extract||11% final, 5.5% in final vesicle solution after mixing with feeding 2|
|5.4μl||Mg-glutamate at 100 mM||(3mM final here) depends on batch|
|3.6μl||K-glutamate at 3M||(60mM final here) depends on batch|
|45μl||AA at 6mM|
Feeding solution 2 for 135μl (with PEG at 6% + RNase A):
|4.05μl||Mg-glutamate at 100 mM||(3mM final here) depends on batch|
|2.7μl||K-glutamate at 3M||(60mM final here) depends on batch|
|33.75μl||AA at 6mM|
|3μl||RNase A at 50X||optional|
|20.25μl||PEG at 40%|
Optional: RNase A (MW = 13700, from Sigma) at 33mg/ml: 2.40 mM
- A final concentration of 50 nM, stock at 2.4 mM is 48000X.
- Stock at 4800X: 10μl + 90μl water/glycerol (50/50). To get stock at 50X: 1μl stock at 4800X + 95μl water
- Place 20μl feeding 1 in a 1.5ml tube
- Add 1μl of reaction into 400μl phospholipid solution, vortex for several seconds with a table top vortex to create an emulsion
- Add 150μl emulsion on top of the 20μl feeding 1
- Wait for the emulsion/feeding 1 interface to stabilize and flatten
- Centrifuge at 11000rpm for 10~15 seconds.
- Remove carefully the mineral oil, take 10μl of feeding 1 (where the vesicles have been formed)
- Add the 10μl vesicle solution formed in feeding 1 + 10μl feeding 2, mix gently (pipet up/down with a new tip)
- Use Frameseal to make a small chamber on a coverslip
- Blot the vesicle solution inside the chamber (try to avoid touching the sticky sides of Frameseal)
- Seal the chamber with a second coverslip; observe on microscope
Sample Images from University of Minnesota
Fig. 1. Image of a 20 uM diameter vesicle observed under 40x objective. The Alpha-hemolysin-eGFP molecules are localized at the lipid membrane.
|Fig. 2a. Brightfield image of vesicles.||Fig. 2b. Fluorescence image of vesicles 2 hours after incubation.||Fig. 2c. Composite image of brightfield and fluorescence images.|