HighPoint/CannonLab:Lipid Vesicle Preparation

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(New page: {{CannonLabMenu}} ==Lipid Film Preparation== ===Materials=== * CHCl<sub>3</sub> * MeOH * dry lipids * screw top glass vial with teflon seal ===Procedure=== # Make 8mL of 3:1 CHCl3:MeO...)
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Revision as of 11:50, 16 February 2012

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Contents

Lipid Film Preparation

Materials

  • CHCl3
  • MeOH
  • dry lipids
  • screw top glass vial with teflon seal

Procedure

  1. Make 8mL of 3:1 CHCl3:MeOH (6mL CHCl3/2mL MeOH)
  2. Add 2.5mL of CHCl3:MeOH solution to 25 mg lipids
  3. Take 1mL (10 mg lipid) of lipid/CHCl3:MeOH solution and place in 4mL vial (2 dram?)
  4. Dry using rotovap
    • Attach vial to rotovap with a rubber septum (silicone septa only work 2-3 times before shrinking)
    • Insert needle through septum to allow solvent to evaporate
    • Evaporation takes <<30 min if conditions are right
  5. Place vial in vacuum desicator overnight to remove remainder of solvent
  6. Close tightly and store aliquot in freezer

Lipid Film Hydration

  • Prepare the buffer appropriate for your experiments
  • Determine the concentration of lipids you wish to use in your experiments
  • Prepare a hydrated lipid suspension much more concentrated than the desired experimental concentration
    • A concentration of 10 mg/mL is often a good starting point
      1. add 1 mL buffer to 10 mg lipid
      2. tape vial to vortexer and shake for 1 h
      3. store hydrated lipids in freezer until you are ready to make LUVs
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