Preparing ssDNA Curtains

Contents

1. Buffers
2. Single-Tethered DNA Curtains

1. Buffers

• Lipids buffer:
  • 10 mM Tris-HCl, pH 7.8
  • 100 mM NaCl
  • Filter through a 0.22 μm syringe filter and store at room temperature. Re-make the buffer every couple of days.
• BSA buffer:
  • 40 mM Tris-HCl pH 8
  • 1 mM DTT
  • 2 mM MgCl2
  • 0.2 mg/ml bovine serum albumin (BSA; fraction VI, Sigma-Aldrich).
  • Filter through a 0.22 μm syringe filter and use the same day as experiment. Do not save buffer overnight.

2. Single-Tethered DNA Curtains

1. Attach a syringe with 10 ml of H2O to one end of the flowcell. Rinse the flowcell with water, while tapping gently. Tapping the flowcell loosens and flushes out all air bubbles within the system. Air bubbles must be avoided, as even a small bubble will ruin the lipid bilayer surface. All subsequent syringes must be attached to the system by making drop-to-drop Luer lock connections.
2. Wash the flowcell with 2–3 ml lipids buffer, contained in a 3 ml Luer lock syringe that is attached to the second nanoport. Alternating between the two flowcell ports reduces the chance of injecting air bubbles into the tubing.
3. Dilute 20-25 μl of Liposome Stock Solution with 975-980 μl of lipids buffer. Inject 1 ml of the diluted liposome solution into the flowcell as a series of three injections with a 5- to 10-min incubation time between injections.
4. Rinse the flowcell with 2–3 ml lipids buffer. Incubate for 30 min to promote vesicle fusion and bilayer growth along the silica surface. If you need to pause, you can leave the flowcell at this point for a few hours.
5. Slowly inject 1-2 ml BSA buffer and incubate for 10 min to allow BSA to block remaining exposed surfaces.
6. Inject 300 μl of 0.1 mg/ml streptavidin in BSA buffer and incubate 10 min.
7. Rinse flowcell with 2–3 ml BSA buffer to flush out free streptavidin. No need to incubate at this point.
8. Inject 100 μl fresh ssDNA mix single-strand-curtains_low_complexity in the flow cell. Slowly inject this solution into the flowcell and incubate 10-20 min depending on desired ssDNA Length.
9. Rinse the DNA out with 2–3 ml BSA buffer. At this point, the flowcell is ready for imaging experiments and should be transferred to the microscope syringe pump system.