During this laboratory you will use agarose gel electrophoresis to
separate DNA fragments which have been generated by digestion of your
plasmid DNA with restriction endonucleases. You will then use a
molecular size marker (a 1 kb ladder) to generate a standard curve of
mobility vs. log bp and use the standard curve to estimate the size
of the fragments separated on the gel (See below for a description of
Gibco-BRL's 1 kb ladder and a picture with the sizes of the marker
A. Casting the gel:
1. Make 25 ml of a 1% (w/v) solution of agarose in TAE buffer.
2. Weigh the container with the mixture and record the mass.
3. Heat the mixture to boiling using the microwave oven. Examine the flask and continue boiling if any agarose is undissolved.
4. Weigh the container with the mixture again and add deionized water to compensate for loss of mass during boiling.
5. Allow the agarose to cool for 3-5 minutes at room temperature before pouring the gel.
6. Make sure the wedges are in place firmly against the ends of the casting tray. Pour all of the agarose solution into the casting tray, being careful not to overflow the tray. Add the comb and leave the gel to cool and solidify.
B. Preparing the samples
1. While the gel is cooling, prepare the DNA samples by adding 1 uL of tracking dye to 5 uL of each restriction digest. The tracking dye is bromphenol blue in a 50% glycerol solution. Adding tracking dye to the sample will increase its density so it falls into the well of the gel and provides a visible marker to monitor the progress of electrophoresis. Also prepare a molecular size standard by mixing 5 uL of the 1 kb ladder with 1 uL of tracking dye.
C. Loading and running the gel
1. Remove the wedges from the casting tray and fill the buffer reservoir with TAE buffer until the buffer is 1-2 mm deep over the gel.
2. Carefully remove the comb by lifting it straight out of the gel slowly.
3. Carefully pipette each mixture (6 uL) into a well in the gel . See the demonstration by the instructor before doing this step. Load one well with the prepared 1 kb ladder.
4. After all the lanes have been loaded, connect the leads from the power supply to the gel box. Make sure the gel is oriented correctly (wells at negative [black] end, DNA will "run to the red"). Set the output level to 100 volts and turn the power on.
5. Run the gel until the tracking dye is approximately 3/4 the way
across the gel.
D. Staining the DNA in the gel with ethidium bromide.
1. After turning the power off, remove the gel from the gel box and submerge it in the ethidium bromide staining solution. WARNING: ETHIDIUM BROMIDE IS A POWERFUL MUTAGEN. USE GLOVES WHEN WORKING WITH IT. Allow the gel to stain for 5 minutes.
2. Remove the gel to a tray of water and allow it to destain for
1- 5 minutes.
1. Place the gel on the transilluminator. Place the clear plastic shield over the transilluminator window before turning on the UV light. WARNING: THE TRANSILLUMINATOR EMITS SHORT WAVE UV LIGHT WHICH WILL DAMAGE SKIN AND EYES, DURING PROLONGED EXPOSURE. BE SURE THAT PROPER SHIELDING IS IN PLACE BEFORE TURNING ON THE TRANSILLUMINATOR.
2. Turn on the transilluminator (BE SURE THAT PROPER SHIELDING IS IN PLACE) and look at the gel. Confirm the presence of DNA (orange bands) and turn off the transilluminator.
3. Remove the plexiglass shield and position the styrofoam shield and camera on the transilluminator. Turn on the transilluminator, wait a few seconds for the light to come on and shoot the picture by squeezing and quickly releasing the trigger on the camera.
4. Turn off the transilluminator. Remove the white tab from the film holder. Then pull out the photograph by grasping the black tab and pulling it completely out of the camera.
5. Wait 30-45 seconds and peel off the print from the backing. Don't touch the inside of the backing which has a photochemical gel on it. Dispose of the backing in the trash can.
The 1 Kb DNA Ladder (U.S. Patent No. 4,403,036) is suitable
for sizing linear double-stranded DNA
fragments from 500 bp to 12 kb. The bands of the ladder each contain from 1 to 12 repeats of a
1018-bp DNA fragment. In addition to these 12 bands, the ladder contains vector DNA fragments that
range from 75 to 1636 bp. The 1636-bp band contains 10% of the mass applied to the gel.