The overall goal of this experiment is to digest fluorescently labeled DNA molecules with methylation and non-methylation sensitive restriction enzymes and determine the digestion rate of the experiment. This method can help answer key questions in the chemistry or biology fields, such as genomics. The advantage of this method is that a scientist can digest pre-stained DNA with an enzyme to determine if the DNA is full-length after the completion of a different experiment.
Demonstrating the procedure will be Drew Thompson, an undergraduate student from my laboratory. To make a 0.7%agarose gel, weigh out 1.4 grams of high gelling temperature agarose in an Erlenmeyer flask. Then add 200 milliliters of 1x TAE buffer and place an inverted beaker on top of the flask.
Heat the solution on a hot plate until bubbles begin to form on the bottom of the flask and float to the top. Remove the solution from the hot plate and let it cool until the flask can be touched with a bare hand. Then pour the gel into a 24.5 centimeter by 21.8 centimeter gel mold with a gel comb to create wells.
Use a pipette tip to pop the bubbles near the comb or on the surface of the gel and allow the gel to solidify for at least 15 minutes. To stain lambda DNA at six different concentrations of dyes, begin by using 1x TE to prepare 300 microliters of 100 nanogram per microliter of lambda DNA. Store the solution at four degrees Celsius.
While preparing a sample for each of the following dye concentrations for each band expected in a restriction digest, stain 75 to 100 nanograms of unmethylated lambda DNA. Incubate the samples at four degrees Celsius overnight. The following day, to determine if a restriction enzyme can digest the stained DNA, add 20 units of restriction enzyme, three microliters of the appropriate buffer with the highest cutting efficient, and distilled autoclaved and filtered water for a 30 microliter reaction.
Place the reactions in a water bath at the optimal enzymatic activity and incubate them for two to four hours. Then stop the reactions by adding two microliters of 0.5 molar EDTA pH 8.0. Remove the sides from the previously prepared gel mold and place the gel into the gel electrophoresis box.
Pour 2.5 liters of 1x TAE buffer into the box. Then carefully remove the gel comb from the gel so the wells are accessible. Pipette the restriction enzyme reactions onto a plastic film, and for each reaction solution, add one microliter of 6x loading dye per five to six microliters of DNA solution.
Then pipette the reactions into the wells. Mix one microliter of one kilobase ladder, nine microliters of 1x TE, and two microliters of 6x loading dye. Then load the solution into the wells that flank the other solutions.
Cover the gel box with a dark blue or black paper or fabric, then connect the gel box to a power supply. Set the power supply to 30 volts and run it overnight. Turn off the lights while the gel is running to prevent photocleavage of the labeled DNA.
The next morning, transfer the gel in the tray to a container with 45 microliters of ethidium bromide and one liter of 1x TAE buffer. Store the container at room temperature in the dark or cover it in foil to prevent exposure to light. Let the gel stay in for at least 45 minutes at room temperature.
To image the gel, place the gel on top of a blue light transilluminator, which emits maximum light output between 400 to 500 nanometers. Take pictures with a camera attached to the station. Determine the digestion rate for the experiment according to the text protocol.
Enzyme efficiency is determined by dividing the total number of expected bands by the number of visible bands. In addition, in order to determine the approximate size of the bands, a control is required to know where the native DNA molecules are expected compared to the stained DNA. The mobility of the bands in lanes three and four has decreased, causing the bands to shift closer to the wells.
In these lanes, the amount of dye does not noticeably affect the mobility, so the bands match the control. Lanes five and six show more bands than the control, which indicates partial digestion, suggesting the dyes affected cleavage at recognition sites. In this experiment, all the expected bands are seen for all dye concentrations, so the dice did not interfere with the digestion of DNA.
To see how the mobility shifted as the concentration of the YOYO-1 dye increased, colored asterisks were inserted next to each band. For example, the 3.5 kilobase bands have red asterisks next to them in each YOYO-1 concentration. The unexpected bands in lanes five and six do not have an asterisk next to the bands.
While attempting this procedure, it's important to remember to cover the gel with a dark piece of paper and complete the running of the gel in a dark room. Also, make sure to use dark tubes for your reactions. After watching this video, you should have a good understanding of how to digest fluorescently-stained DNA molecules with restriction enzymes and determine the digestion rate of the reaction.
Don't forget that working with ethidium bromide can be extremely hazardous, so precautions such as wearing gloves and safety glasses should be taken while performing the procedure. Also, make sure to wear appropriate safety glasses when using a transilluminator.
