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Vesicle Preparation

• 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.

1. 10mg + 1ml mineral oil, vortex strongly, heat at 50C for 10 minutes.

2. Prepare a sub stock at 2mg/ml (200μl at 10mg/ml + 800μl mineral oil).

3. Heat at 50C for 1 hour.

4. Incubate at RT for 6 hours.

Reaction composition for 90μl (100% volume):

Volume	Contents	Note
30μl	extract
6.5μl	3-PGA buffer
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)
10.1μl	water

Feeding solution 1 for 180μl (no PEG + extract):

• Note: PEG interferes with vesicle formation!

Volume	Contents	Note
40μl	S30 buffer
20μl	extract	11% final, 5.5% in final vesicle solution after mixing with feeding 2
13μl	3-PGA buffer
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
53μl	water

Feeding solution 2 for 135μl (with PEG at 6%, + RNase A):

Volume	Contents	Note
45μl	S30 buffer
9.75μl	3-PGC buffer
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%
16.5μl	water

• 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

Vesicle formation (takes only a few min):

• 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