User:Emmalee Jones/Notebook/Lab Notebook/2010/02/19

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More CBME

  • Extruded egg PC lipid solution following technique from online at the manufacturer's website + Dr. Chi's notes + the guy who was here before me's notes. Before beginning, I went to CRLS and got 1 lb of dry ice for the CO2 bath later today


Basic Procedure for Extrusion with problems listed at the end

  • 1. Rehydrate lipid by preparing a 2mM lipid and buffer solution. For 5 mg of egg PC, this is 3289 μL of buffer.
  • 2. Keep solution above transition temperature, but in my case with Egg PC room temperature is fine
  • 3. Vortex for 30 minutes at a low setting - should appear foamy
  • 4. Prepare CO2 & ethanol bath (sidenote: when finished, ethanol can be transferred into an empty bottle labeled "dirty ethanol" and used again)
  • 5. Heat up water in a beaker on hot plate to 65°C
  • 6. Freeze vial until bottom of frozen cake cracks (use tweezers to hold vial)
  • 7. Thaw in hot water bath until cake melts
  • 8. Repeat for 5 cycles
  • 9. Clean all extruder parts with Milli-Q water, than ethanol, than Milli-Q again
  • 10. Dry parts with Kimwipes
  • 11. Assemble the mini-extruder
   i.    Follow pictures from instructions on the Avanti site at 
         http://avantilipids.com/index.php?option=com_content&view=article&id=532&Itemid=296
   ii.   Place 2 internal membrane supports on flat surface with O-rings facing up
   iii.  Prewet 2 filter supports with Milli-Q water (these are really thin, be careful not 
         to take more than one out from the package by mistake) and place over internal membrane
         support inside O-ring inner diameter
   iv.   Insert internal membrane support into extruder outer casing with O-ring facing up
   v.    Place 1 Polycarbonate membrane in extruder outer casing over filter support and O-ring 
         (the membranes are clear and shiny, don't take the blue filter papers that they come 
         packed in by mistake)
   vi.   Pre-wet two more filter supports with Milli-Q water and place over the other internal
         membrane support
   vii.  Carefully place second internal membrane support into casing, O-ring facing down and 
         careful not to lose the filter supports or twist when it comes in contact with the 
         membrane
   viii. Place teflon bearing (thin circle of teflon so things will fit tight) into retainer nut 
   ix.   Place retainer nut on threaded end of extruder outer casing and tighten by hand 
         until finger tight
  • 12. Load sample into one of the gas-tight syringes (which has been cleaned with ethanol, Milli-Q water,and then prewet with buffer) and carefully place into one end of the mini-extruder
  • 13. Place empty syringe into other end of the mini-extruder
  • 14. Insert apparatus into extruder stand, you have to apply a little force to get it in, making sure that the apex of the hex nut is pointing up and down (from above it should look pointed not flat)
  • 15. Push filled syringe plunger until solution completely transferred, then push other syringe, etc.
  • 16. Extrude 19 times (always finish on an odd number so that the final extrusion fills the alternate syringe)
  • 17. Remove syringe, clean parts again
  • 18. Store vesicles in 4°C fridge
  • Had a few problems that probably compromised integrity of sample, but continued since it was good to practice the method if nothing else.
  • 1st problem - Desiccator took two days instead of one
    Possible solution - When I asked her, Dr. Chi remembered that when they did this before, they 
    actually did it first by blowing the vial dry with nitrogen or filtered air to get most of the 
    chloroform evaporated, and then they left it overnight in the desiccator
  • 2nd problem - Made the solution 2 mM with PBS which I made last semester (still looked ok). For Egg PC Mol Weight = 760.09 g/mol, so for 5 mg that meant 3289 μL to make it 2 mM - which fit fine in my 4 mL vial. Now the problem was that the rack on the vortex spinner is designed to be used with 2 mL tubes, so the vial is too big, I couldn't tape it well enough to get it to stay on very well.
    Possible solutions include finding a bigger vial rack (unlikely), holding the vial physically 
   (for 30 min this would be excruciating), or finding better tape / better top for the spinner to 
    tape to
  • Sidenote: Hotplate takes FOREVER to heat, turn it on at the beginning of the 30 min vortex spinning
  • 3rd problem - Syringes for the extruder have a really short needle and do not fit into the 4 mL vial. I had to put it in a 10 mL vial, but I wasn't sure how well it transferred. Of course, there would be NO WAY the 10 mL vial would fit on the vortex spinner rack.
    Possible solution is to prepare in 10 mL vial initially, jury-rig something for the vortex 
    spinner and after extruding put in a 4 mL vial



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