The overall goal of this experiment is to visualize the energy transfer from one dye modified DNA strand to another. This method can help on the key questions in the field of molecular biology. Such as RNA interference by monitoring siRNA processing and integrity in living cells in real time.
The main advantage of this technique, is the ease of monitoring the florescence reach out and the color change, giving a clear result. So false positive results are eliminated. The begin this procedure, add the appropriate reagents as indicated in the text protocol to a 20 milliliter, round bottom flask, equipped with a magnetic stir bar and reflux condenser.
Then, add zero point zero six milliliters of piperidine. And heat the reaction mixture to 80 degrees celsius for four hours. After the reaction has cooled to room temperature, collect the resulting precipitate by filtration.
And wash with diethyl ether three times. Next, add one hundred milliliters of diethyl ether to the supernatant. And collect the resulting precipitate by filtration.
After washing three times with diethyl ether, combine the precipitate with isolated solid from the first filtration. Using the computer, connected to a DNA synthesizer, enter the desired DNA sequence and coupling method. Following the prompts from the manufacturer's protocol.
Next, mount the column containing the controlled pour glass, that is modified with one micro mole of the first face into the synthesizer. Now, start the synthesis on the DNA synthesizer. Following workup of the DNA strands, add the appropriate regents as indicated in the text protocol to the diethylized alkyne, modified DNA sample.
Incubate the sample at 60 degrees celsius for one point five hours. Then, transfer the sample to a 10 milliliter tube. After cooling to room temperature, add 150 microliters, of a zero point zero five molar EDTA disodium salt solution, and 450 microliters of a zero point three molar sodium acetate solution to the DNA sample.
Following this, add 10 milliliters of ethanol to the sample. And keep it at minus 32 degrees celsius for 16 hours. Next, centrifuge the sample for 15 minutes at one thousand times G.When finished, remove the supernatant.
Wash the resulting DNA pellet with two milliliters of cold 80%ethanol. Then dry the pellet under reduced pressure using a freeze drier. Purify the crude DNA pellet by HP LC.Monitor the run by checking 260 nanometers, and 459 nanometers for Dye one.
Or 542 nanometers for Dye two. Collect the fractions that show absorption in both wave length channels. For single strand absorption spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate buffer.
Next, transfer the previously prepared DNA1 solution into a one centimeter, quartz glass cuvette. Record the absorption. Then determine the maximum value of dye absorption.
For single strand fluorescent spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate, using the excitation wave length of Dye one. Following this, place the cuvette with the DNA1 solution into the spectrometer. Record the florescence spectrum.
To perform the double strand, fluorescent spectroscopy measurement, place the cuvette with the double strand of DNA1 and DNA2 solution into the spectrometer. Then record the florescent spectrum using the excitation wave length of Dye one. To gain a better understanding of what the recorded spectra are showing, irradiate the cuvettes with hand held UV lamps.
Observe the change in florescence color from the single to the double stranded DNA. The recorded absorption spectra show the maxima at 465 nanometers for single stranded DNA1 and 546 nanometers for single stranded DNA2. The annealed double strand of DNA1 and DNA2 shows maxima at both 469 and 567 nanometers.
Both absorption maxima show a bathaohromic shift. And the smallest spectroscopic changes are the result of excitonic interactions between the dyes within the double helical DNA architecture. The corresponding fluorescent spectra exhibit a maxima at 537 nanometers for single stranded DNA1.
At 607 nanometers for single stranded DNA2. And at 615 nanometers for annealed, double strand of DNA1 and DNA2. Double stranded of DNA1 and DNA2, shows solely the emission maximum of Dye two, although Dye one is selectively excited, which evidences that the energy transfer occurs efficiently from Dye one to Dye two.
After watching this video, you should have a good understanding of how to post synthetically modify DNA by a couple catalytic reaction of an alkyne labeled DNA strand with an A side.
