To begin, arrange the freshly prepared filter sterilized blocking buffer, 10X reaction buffer one and two on a working platform. Place open tubes containing 2.5 milliliters of blocking buffer and five milliliters of ultrapure water in a desiccator and degas under vacuum. After 15 minutes, release the pressure and remove the tubes.
Place the prepared biotin-PEG flow cell assembly on a microscope stage and secure it using adhesive putty at each end. Connect the flow cell outlet tubing to the syringe pump via needle. Switch on the objective heater to 30 degrees Celsius.
Secure one to two milliliters of degassed water in a tube to a separate piece of adhesive putty near the flow cell. Insert the inlet tubing into the tube until it reaches the bottom and flow water through the channel. Increase the flow rate to remove any trapped bubbles near the inlet tubing.
Add 100 microliters of degassed blocking buffer to a 20 microliter aliquot of one milligram per milliliter streptavidin. Attach the open tube to adhesive putty. Transfer the inlet tubing from the water to the streptavidin and start the flow at a rate of 40 microliters per minute for two minutes.
After five minutes, wash out excess streptavidin with the blocking buffer. Flow in biotinylated DNA diluted in blocking buffer containing 25 nanomolar SYTOX Orange. Image using live view with the 532 nanometer laser to watch the DNA tethering to the surface in real time.
Once the desired density of DNA on the surface is achieved, flow in a blocking buffer containing 25 nanomolar SYTOX to wash out free DNA. Now, flow and biotinylated anti-digoxigenin antibody diluted in blocking buffer containing 25 nanomolar SYTOX Orange. Wash out the biotinylated anti-digoxigenin antibody and SYTOX Orange with blocking buffer.
Flow 50 microliters of ATP-gamma-S mix into the flow cell. Add purified CMG helicase to approximately 100 nanomolar final concentrations in 30 microliters of ATP-gamma-S mix and flow at 20 microliters per minute for 20 microliters. Incubate for 15 minutes.
Next, flow in ATP RPA mix at 40 microliters per minute for 80 microliters. Using each laser with 50 to 100 milliseconds exposure, visualize and acquire EGFP RPA with a 488 nanometer laser, then acquire CMG with a 640 nanometer laser. Finally, visualize and acquire SYTOX Orange stained DNA with a 532 nanometer laser.
CMG helicase unwinding of DNA was visualized by the linear growth of the yellow RPA tracked over time, indicating the unwound DNA strands. Single-stranded DNA damage reduced the number of successful unwinding events observed as demonstrated by fewer complete traces in the data.
