This method can help answer the key questions in the field of single molecule imaging of DNA protein interactions such as DNA replication, gene repair and the maintenance of chromosome structure. The main advantage of this technique is that one can obtain modified lambda DNA high collagen and then rapidly cure the labeled proteins. Visual demonstration of this method is critical because the flow cell assembly steps are difficult to learn.
To clean the coverslips, place 20 coverslips into four staining jars, pour ethanol into them and sonicate for 30 minutes in ethanol. Then rinse the coverslips with ultrapure water three times. Next, sonicate the coverslips with one molar potassium hydroxide for 30 minutes and rinse the coverslips with ultrapure water three times.
Then repeat the ethanol and potassium hydroxide sonication once. Sonicate the coverslips with acetone for 30 minutes and then thoroughly rinse with ultrapure water. Now place the coverslips into Piranha solution and incubate at 95 degrees Celsius for one hour.
Following incubation, rinse the coverslips with ultrapure water five times. Then wash each coverslip extensively with ultrapure water. Use paper to dry the coverslip from its edge and place the coverslip into a staining jar.
Thoroughly dry the coverslip in a 110 degree Celsius oven. Now rinse the coverslip with methanol and then place the staining jar back into the 110 degree Celsius oven to dry the coverslip. To perform coverslip functionalization, add 70 milliliters of Silane solution into each jar, screw the cap and leave the jar at room temperature overnight.
Wash the coverslips using three beakers filled with ultrapure water. Dry the coverslips thoroughly using nitrogen gas. Dissolve 150 milligrams of methoxy-PEG and six milligrams of biotin-PEG into one milliliter of freshly prepared 0.1 molar sodium bicarbonate.
Centrifuge at 17, 000 times g for one minute to remove insoluble PEGs. Now pipette 100 microliters of PEG solution on the center of the silanized coverslip and place another silanized coverslip on the top. Incubate the coverslips with PEG solution for at least three hours in the dark.
Separate the coverslip pairs and keep the functionalized surface face up. Extensively rinse the coverslips using ultrapure water and dry them with nitrogen gas. Now mark the functionalized side of the coverslips on one of the corners using a marker pen.
Place one coverslip into a 50 milliliter tube drilled with one hole on its cap. Place the tube into a plastic bag, seal the bag using a vacuum sealer and store it at minus 20 degrees Celsius. To assemble the flow cell, cut a channel at the center of a piece of double-sided tape using a puncher.
Peel off the paper side of the double-sided tape and paste it on a glass side with two holes. It is easier to remove air bubbles by peeling off the paper side than the plastic side of the double-sided tape. Press the tape to remove air bubbles.
Then cut a functionalized coverslip into four pieces using a diamond-tipped glass scribe and remove the debris using nitrogen gas keeping the functionalized side up. Peel off the plastic side of the double-sided tape and paste the slide on the functionalized coverslip. Press gently to remove air bubbles between the coverslip and the tape.
Removing air bubbles thoroughly can protect the flow cell from leaking while buffers are pumped into it. Now insert the inlet tubing into the small hole and insert the outlet tubing into the big hole. Fix the tubing using epoxy.
Manually pump 20 microliters of 0.2 milligrams per milliliter Streptavidin into the flow cell using a syringe and incubate at room temperature for 10 minutes. Then pump blocking buffer into the flow cell to replace Streptavidin and keep it at room temperature. Obtain the focal alignment of red and far red with A5 on the fluorescence microscope test slide number one by simultaneous excitation using a 532 nanometer laser and a 640 nanometer laser.
Produce dual wavelength images with splitting optics. Place the flow cell on the microscope and connect its outlet tubing to a longer tubing that is connected to a 10 milliliter spring installed on an automated infusion withdrawal programmable pump. Remove air bubbles in the flow cell by injecting blocking buffer and flipping the outlet tubing.
Add 0.5 microliters of biotinylated lambda-ARS317 DNA into 80 microliters of blocking buffer. Pump the prepared mixture into the flow cell at 25 microliters per minute for two minutes. Then flush the lambda-ARS317 DNA using 200 microliters of blocking buffer at a rate of 50 microliters per minute.
Now pump 200 microliters of binding buffer at a rate of 50 microliters per minute to remove the blocking buffer in the flow cell. Next, add two microliters of ORC-Qdot705, one microliter of DTT, and one microliter of ATP into 96 microliters of binding buffer. The final concentration of ORC-Qdot705 is 0.2 nanomolar.
After pumping the binding buffer as detailed in the text protocol, pump 20 microliters of the prepared ORC-Qdot705 solution at a rate of 10 microliters per minute into the flow cell. Flush out excessive ORC-Qdot705 using 200 microliters of binding buffer at a rate of 100 microliters per minute. Then pump binding buffer with 30 nanomolar SYTOX Orange into the flow cell to stain DNA substrates at a rate of 100 microliters per minute.
Finally, excite the ORC-Qdot705 signal using a 405 nanometer laser and excite the SYTOX Orange stained DNA signal using a 532 nanometer laser. Observe the signals simultaneously with 100 microliters per minute flow using a quad band bandpass filter. Record the images by EMCCD with 100 milliseconds per frame.
The illustration of lambda-ARS317 DNA substrate is shown here. Qdot705-labeled ORC was excited by a 405 nanometer laser. SYTOX Orange stained DNA was excited by a 532 nanometer laser.
The merged result suggests that Qdot705-labeled ORC binds at ARS317. The result of ORC-binding distribution on lambda-ARS317 DNA shows that ORC binds at ARS317 with a high abundance. Following this procedure, other methods like single-molecule FRET can be performed in order to answer additional questions regarding protein-protein interactions and protein dynamics.
After its development, this technique paved the way for researchers in the field of single-molecule fluorescent imaging to explore DNA protein interactions in reconstituted systems of DNA replication and DNA repair in vitro. Don't forget that working with Piranha solution can be extremely hazardous and the precautions such as wearing of protective face masks should always be taken while performing this procedure.
