Making bilayers

1. Cleaning coverslips
   1. Heat a 20% (v/v) solution of 7x detergent (www.mpbio.com) on a hot plate until solution is clear, typically solution is 90 °C. WARNING: Do not leave unattended!
   2. Place coverslips (25×25, #1) in a ceramic holder
   3. Immerse coverslips (25×25, #1) in a ceramic holder into solution for 5 minutes
   4. Rinse extensively with ddH₂O.
   5. Dry cover slips with N₂ gas. Place in container and cover with foil.
   6. Plasma-etch coverslips in UV-ozone chamber (located on 4^th floor) for 5 minutes.
2. Extruding vesicles
   1. Aliquot 5 mg of a 1% (mol/mol) DHPE-TR:POPC into 20 mL scintillation glass vile.
   2. Dry out chloroform using using roto-vap (ask around to be trained on the instrument).
   3. Re-suspend vesicles in 0.5 mL ddH₂O to a final concentration of 10 mg/mL.
   4. Extrude vesicles through 100 nm pore size filter 20 times. Solution should look translucent afterwards.
3. Making flow cell
   1. Using tweezers, remove peel from Secure Seal Hybridization chamber gasket (Invitrogen, S24720, Lot#_______)
   2. Place a clean coverslip on the adhesive side and gently press them together.
4. Making bilayers
   1. Make “vesicle solution”
   2. Make flow cell using clean coverslips. Add “vesicle solution” to flow cell and incubate for several minutes.
   3. Rinse flow cell in 0.5 L of ddH₂O. Repeat 3 times with fresh water each time.
   4. Fill flow cell with reaction and seal with Adhesive seal tabs (Invitrogen, A18211, Lot#______).

Vesicle Solution (0.5 mL)

10 mM TrisHCl pH 7.2            → 5 µL of 1 M

100 mM NaCl                         → 10 µL of 5 M

1 mg/mL POPC vesicles         → 50 µL of 10 mg/mL