- Cleaning coverslips
- 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!
- Place coverslips (25×25, #1) in a ceramic holder
- Immerse coverslips (25×25, #1) in a ceramic holder into solution for 5 minutes
- Rinse extensively with ddH2O.
- Dry cover slips with N2 gas. Place in container and cover with foil.
- Plasma-etch coverslips in UV-ozone chamber (located on 4th floor) for 5 minutes.
- Extruding vesicles
- Aliquot 5 mg of a 1% (mol/mol) DHPE-TR:POPC into 20 mL scintillation glass vile.
- Dry out chloroform using using roto-vap (ask around to be trained on the instrument).
- Re-suspend vesicles in 0.5 mL ddH2O to a final concentration of 10 mg/mL.
- Extrude vesicles through 100 nm pore size filter 20 times. Solution should look translucent afterwards.
- Making flow cell
- Using tweezers, remove peel from Secure Seal Hybridization chamber gasket (Invitrogen, S24720, Lot#_______)
- Place a clean coverslip on the adhesive side and gently press them together.
- Making bilayers
- Make “vesicle solution”
- Make flow cell using clean coverslips. Add “vesicle solution” to flow cell and incubate for several minutes.
- Rinse flow cell in 0.5 L of ddH2O. Repeat 3 times with fresh water each time.
- 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