The overall goal of this procedure, is to make a convenient DNA analysis system for an epifluorescent microscope, to manipulate single molecule DNA on modified glass surfaces. This method can help answer key questions in DNA visualization field. When examining DNA self, as well as DNA binding proteins and other small molecules.
The main advantage of this technique is the ability to observe and analyze moving DNA with ease. To begin, place glass coverslips on a PTFE rack and secure them in place, using a piece of PTFE thread seal tape. After wrapping the coverslips, leave a five centimeter long piece of the tape to be used for handling the rack during the cleaning procedure.
In a fume hood, combine 350 millileters of sulfuric acid and 150 milliliters of hydrogen peroxide in a one liter glass beaker to make a piranha etching solution. Then, place the rack of coverslips into the solution. After two hours, empty the beaker and rinse the coverslips thoroughly with deionized water.
Continue rinsing the coverslips until the PH of water in the beaker, reaches a neutral PH.Then, sonicate the racks of coverslips in a beaker contaning water for 30 minutes, to clean and derivative the glass substrates. Empty water from the beaker, just before aminosilanization. To begin, prepare the aminosilanization solution, by adding two milliliters of alkoxysilane, and 10 milliliters glacial acetic acid to 200 milliliters of methyl alcohol in a clean, polypropylene container.
Followed by placing the clean coverslips into the solution. Then, place the cleaned coverslips in the prepared solution and shake them at room temperature and 100 rpm's for 30 minutes. Next, sonicate the container with the derivatized coverslips for 15 minutes at 75 watts.
Then, shake them again at room temperature and 100 rpm's for at least 30 minutes. Once finished, empty the solution from the beaker and rinse the coverslips three times, carefully. Once with methyl alcohol and twice with ethyl alcohol.
Following the final rinse, store the coverslips in ethyl alcohol and use them within two weeks. First, make 10 milliliters of a 0.1 molars sodium bicarbonate solution and filter it through a 0.22 micron syringe filter. Then, dissolve two milligrams of biotin PEG succinimidyl carbonate and 80 milligrams of PEG succinimidyl valerate into 350 micro liters of the filtered sodium bicarbonate solution.
Protect the solution from light using a light protection tube. Vortex the tube vigorously, for 10 seconds and then, centrifuge it at 10, 000 x g for one minute to remove any bubbles. Next, rinse a glass slide with acetone followed by ethyl alcohol.
Once rinsed, allow the slide to air dry completely. Place a 50 microliter drop of the PEG solution onto the clean, glass slide. Slowly lower an amino silanized coverslip onto the droplet at a slight angle, so as to not generate any air bubbles under the coverslip.
Then, place the slides into a level, dark and humidified chamber for at least three hours to overnight, at room temperature. When finished, seal the residual PEG solution in the light protection tube, and keep it at four degrees Celsius. Following incubation, rinse the PEGylated coverslips thoroughly with deionized water and then store them in a dark and dry place until they are used.
In order to create the flow chamber, place double-sided, sticky tape strips on an acrylic holder, so that it is aligned perpendicularly to inlet and outlet holes and does not perturb any of the channel holes. Then, rub the tape, using a pipet tip, to seal the channels. Next, place a PEGylated coverslip, with the PEGylated side face down, on the top of the tape to make flow chambers.
To prevent any solution from leaking, press the top of a coverslip over the area where double-sided tape is placed. Then, add a quick dry, epoxy glue to the edges of the chambers, to secure it in place. Next, connect a 2.5 centimeter long piece of tubing to a gas tight syringe and seal the joint with epoxy glue.
Then, connect a long, flexible tube to the tubing coming from the syringe. And again, seal the joint with epoxy glue. Fill the tubing that is linked to the syringe with deionized water, making sure there are no air bubbles.
Next, insert the tubing into the hole of the flow chamber that is sealed with epoxy glue. And place a 200 microliter pipet tip on the other hole, to be used as a reservoir. Set the flow rate of a syringe pump to 50 microliters per minute.
Then, flow 20 microliters of an avidin protein solution into the chamber and keep it there for 10 minutes. Next, load 20 microliters of biotinylated oligo deoxy nucleotides, into a syringe. Flow it into the chamber, and keep it there for 10 minutes.
If a terminal transferase is used, skip the loading of oligo deoxy nucleotides. Then, load 20 microliters of a DNA solution into a syringe, flow it into the chamber at a rate of 10 microliters per minute and keep it there for 30 minutes. Following the 30 minute incubation, wash the flow chamber with 40 microliters of 1X TE buffer at 50 microliters per minute.
Once washed, load the chamber with 40 microliters of the same FP-DBP at a concentration of 80 nano-molar, in the 1X TE.To see the full stretch range of DNA molecules, apply different flow rates according to the DNA lengths used. Observe the DNA under a 60X objective lens, using a fluorescent microscope. Here is a fluorescent microscope video showing bacteriophage lambda DNA molecules, tethered to the surface, being stretched by flow after ligation with biotinylated complimentary oligo nucleotides.
Under flow, the fully elongated DNA molecules, measure 16.5 microns in length. In contrast, the bacteriophage T4 DNA momlecules, elongated to a length of 56.4 microns, under flow. This elongation is reversible, and the DNA can be extended and relaxed multiple times.
After watching this video, you should have a good understanding of how to use FP-DBP in larger DNA analysis as well as demonstrate stretching and relaxation of DNA polymer.
