Biomolecular Breadboards:Protocols:Vesicles

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m (New page: {{Template:Biomolecular Breadboards}} ===Vesicle Preparation=== *Protocol by Vincent Noireaux, University of Minnesota, School of Physics and Astronomy '''Phospholipid preparation: eggP...)
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'''Phospholipid preparation: eggPC (Avanti 840051P, MW = 770)'''  
'''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.
*Note: Special order phospholipids in small aliquots (~25 mg), as the lipids in the bottle do not last long after opening.
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1. 10mg + 1ml mineral oil, vortex strongly, heat at 50C for 10 minutes.
 
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2. Prepare a sub stock at 2mg/ml (200μl at 10mg/ml + 800μl mineral oil).  
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* 10mg + 1ml mineral oil, vortex strongly, heat at 50C for 10 minutes.
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* Prepare a sub stock at 2mg/ml (200μl at 10mg/ml + 800μl mineral oil).  
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3. Heat at 50C for 1 hour.   
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* Heat at 50C for 1 hour.   
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* Incubate at RT for 6 hours.
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4. Incubate at RT for 6 hours.
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'''Reaction composition for 90μl (100% volume):'''
'''Reaction composition for 90μl (100% volume):'''
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{| class="wikitable"
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{| border="3" cellpadding="5"
|-
|-
! Volume !! Contents !! Note  
! Volume !! Contents !! Note  
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'''Feeding solution 1 for 180μl  (no PEG + extract):'''
 
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*Note: PEG interferes with vesicle formation!
 
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{| class="wikitable"
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'''Feeding solution 1 for 180μl  (no PEG + extract):''' PEG interferes with vesicle formation
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{| border="3" cellpadding="5"
|-
|-
! Volume !! Contents !! Note  
! Volume !! Contents !! Note  
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'''Feeding solution 2 for 135μl (with PEG at 6%, + RNase A):'''
 
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{| class="wikitable"
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'''Feeding solution 2 for 135μl (with PEG at 6% + RNase A):'''
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{| border="3" cellpadding="5"
|-
|-
! Volume !! Contents !! Note  
! Volume !! Contents !! Note  
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* Optional: RNase A (MW = 13700, from Sigma) at 33mg/ml: 2.40 mM.
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'''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.
* 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
* 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):
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'''Vesicle formation:'''
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* place 20μl feeding 1 in a 1.5ml tube
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* Place 20μl feeding 1 in a 1.5ml tube
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* add 1μl of reaction into 400μl phospholipid solution, vortex for several seconds with a table top vortex to create an emulsion
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* Add 1μl of reaction into 400μl phospholipid solution, vortex for several seconds with a table top vortex to create an emulsion
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* add 150μl emulsion on top of the 20μl feeding 1.
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* Add 150μl emulsion on top of the 20μl feeding 1
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* wait for the emulsion/feeding 1 interface to stabilize and flatten
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* Wait for the emulsion/feeding 1 interface to stabilize and flatten
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* centrifuge at 11000rpm for 10~15 seconds.
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* Centrifuge at 11000rpm for 10~15 seconds.
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* remove carefully the mineral oil, take 10μl of feeding 1 (where the vesicles have been formed).
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* Remove carefully the mineral oil, take 10μl of feeding 1 (where the vesicles have been formed)
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* add the 10μl vesicle solution formed in feeding 1 + 10μl feeding 2, mix gently (pipet up/down with a new tip)
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* Add the 10μl vesicle solution formed in feeding 1 + 10μl feeding 2, mix gently (pipet up/down with a new tip)
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* use Frameseal to make a small chamber on a coverslip
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* Use Frameseal to make a small chamber on a coverslip
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* blot the vesicle solution inside the chamber (try to avoid touching the sticky sides of Frameseal)
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* Blot the vesicle solution inside the chamber (try to avoid touching the sticky sides of Frameseal)
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* seal the chamber with a second coverslip; observe on microscope
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* Seal the chamber with a second coverslip; observe on microscope

Revision as of 19:25, 2 August 2013

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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.
  • 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):

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): 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:

  • 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
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