1. DNA Biotinylation

1. Biotinylation of blunt ended DNA using Terminal Transferase (TdT)
   Note: Use T4 DNA (166 kbp), which is a blunt ended DNA.
   1. Add 5 µl of 2.5 mM CoCl₂, 5 µl of 10× reaction buffer, 0.5 µl of 10 mM biotin-11-dUTP, 0.5 µl of terminal transferase (10 units), and 0.5 µl of T4 DNA (0.5 µg/µl) to the reaction mixture. Make to a final volume of 50 µl by adding 38.5 µl of water.
   2. Incubate the reaction mixture at 37 °C for 1 hr.
      Note: Extended reaction time may yield double-tethered DNA, i.e., it is possible that biotins are tagged at both ends.
   3. Stop the reaction by adding 5 µl of 0.5 M EDTA, pH 8.
   4. Keep the tube at 4 °C.
2. Biotinylation of sticky ended DNA Using DNA Ligase
   Note: Use λ DNA (48.5 kbp), which is a sticky-end DNA.
   1. Add 1 µl of T4 DNA ligase, 5 µl of 10× ligation buffer, 1 µl of 25 ng/µl λ phage DNA, and add 43 µl of water to make a final volume of 50 µl.
   2. Keep the tube at 4 °C.

2. Functionalized Surface Derivatization

Note: In order to tether an end of a DNA molecule on glass surface, a primary amine group is silanized on a coverslip followed by biotin-PEG coating as shown in Figure 1. This PEGylation process is important for single-molecule DNA imaging because it can significantly decrease random noise created by attachment of undesired molecules on the surface.

1. Piranha Cleaning
   Note: Piranha solutions react vigorously with organic materials, therefore it should be handled with care, following proper safety guidelines.
   1. Place coverslips on a polytetrafluoroethylene (PTFE) rack, and hold them by PTFE thread seal tape lengthwise, half on the edge of the surfaces and half on the rack. After wrapping, leave a long piece (~ 5 cm) of tape for handling the rack during the cleaning procedure.
   2. Fill a 1 L glass beaker with 350 ml of H₂SO₄ and 150 ml of H₂O₂ to make piranha solution in a fume hood. Place the rack in piranha solution for 2 hr.
   3. Empty piranha solution from the beaker, and rinse coverslips thoroughly with deionized water until the pH of water in the beaker reaches neutral. Use pH paper strips.
   4. Sonicate the racks of cover slips in the beaker containing water, for 30 min. Empty water from the beaker just before amino silanization. Use 75 W of sonication power to clean and derivatize the glass substrates.
2. Aminosilanization on Glass Surface
   1. Add 2 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine and 10 ml of glacial acetic acid to 200 ml of methyl alcohol in a clean polypropylene container for preparing the aminosilanization solution.
   2. Place cleaned coverslips in the polypropylene container. Shake them at 100 rpm and room temperature for 30 min.
   3. Sonicate the beaker with derivatized cover slips for 15 min at 75 W. Shake them at 100 rpm and room temperature for at least 30 min.
   4. Empty the solution from the beaker, and rinse coverslips carefully, once with methyl alcohol, and twice with ethyl alcohol. Store coverslips in ethyl alcohol and use them within two weeks.
3. PEGylation of the coverslip
   1. Make 10 ml of 0.1 M sodium bicarbonate (NaHCO₃). Filter the solution with a 0.22-µm syringe filter.
   2. Dissolve 2 mg of biotin-PEG-succinimidyl carbonate (biotin-PEG-SC) and 80 mg of PEG-succinimidyl valerate (mPEG-SVG) in 350 µl of NaHCO₃. Use a light protection tube.
   3. Vortex the tube vigorously for 10 sec, and centrifuge it at 10,000 × g for 1 min to remove bubbles.
   4. Rinse a glass slide with acetone, followed by rinsing with ethyl alcohol. Allow the slide to air dry completely.
   5. Place a drop (50 µl) of PEG solution on the clean glass slide. Cover the droplet gently with an amino silanized cover slip, without generating any bubbles.
   6. Place slides for 3 hr to overnight in a dark, well-leveled humid chamber at room temperature. Use an empty pipette tip holder as the chamber, with water at the bottom. Seal residual PEG solution in the tube and keep it at 4 °C.
   7. Rinse the PEGylated coverslip thoroughly with deionized water. Store it in a dark and dry place until use.

3. Assembling a Flow Chamber

1. Fabricate a punched acrylic holder, as in Figure 2. Ensure that the diameter of the tubing insert is 0.762 mm, and designate one hole is an inlet and the other is an outlet (Figure 2).
2. Place double-sided sticky tape strips (width 5-6 mm) on an acrylic holder (Figure 2), aligning perpendicularly to an inlet and outlet hole, not perturbing any channel holes. Use these tape strips as multi-channel walls to make chambers. Scrub the tape using a pipet tip.
3. Place a PEGylated coverslip on the top to make flow chambers, with the PEGylated side facedown.
4. To prevent any solution from leaking, press the top of a coverslip over the area where double-sided tapes are placed. Add quick-dry epoxy glue to close the edges of the chamber at the top and at the bottom (yellow color in Figure 2).
5. Connect a short piece (2.5 cm) of tubing (outer diameter, OD, 0.042") to a gas-tight syringe and seal the joint with epoxy glue.
6. Connect a long flexible tubing (OD: 0.03") to the tubing linked to the syringe and seal the joint with epoxy glue.
7. Fill the tubing linked to the syringe with DI water. Make sure there is no air bubble.
8. Insert the tubing into the hole of the flow chamber sealed with epoxy glue.
9. Place a yellow tip (200 µl tip) on the other hole as a reservoir.

4. Sample Loading into Flow Chamber

Note: Neutravidin can be replaced with other avidin proteins such as streptavidin. All of the reactions can be performed at room temperature, unless it is mentioned. Take sample solution in a yellow tip, and install the tip with the solution onto the hole of acrylic holder (Figure 2b).

1. Set the flow rate of the syringe pump at 50 µl/min. Load 20 µl of avidin protein (25 µg/ml in T50 solution, Tris 10 mM, NaCl 50 mM, pH 8), and keep it for 10 min.
2. Load 20 µl of biotinylated oligodeoxynucleotides (100 µM in 1× TE), and keep it for 10 min. If terminal transferase is used, skip the loading of oligodeoxynucleotides.
3. Load 20 µl of DNA solution from Step 1 into the flow chamber at a flow rate of 10 µl/min, and keep it for 30 min.
4. Wash the flow chamber with 1× TE, and load 40 µl of FP-DBP¹⁰ (~80 nM).
5. Observe DNA under a fluorescent microscope with continuous flow of staining molecules in 1× TE on a 60X objective lens. Use 488 nm solid state laser for excitation of FP(eGFP)-DBP. For full stretch of DNA molecules, apply different flow rates according to the DNA lengths used. For example, apply 50 µl/min for 48.5 kbp of λ DNA, and 100 µl/min for 166 kbp of T4 DNA.
   1. For recycling of the acrylic holder, soak assembled flow chambers in a household detergent solution¹². Remove tapes and epoxy with a razor blade and rub with hands to completely take them off. Unclog holes with syringe needles. Keep the holders in deionized water until further use.