During this laboratory you will use agarose gel electrophoresis to
separate DNA fragments which have been generated by PCR. You will
then use a molecular size marker (a 1 kb plus ladder) to
estimate the size of the fragments separated on the gel (See below
for a description of Gibco-BRL's 1 kb plus ladder and a picture with
the sizes of the marker fragments)
A. Casting the gel:
1. Make 25 ml of a 2% (1% = 1g/100 ml, w/v,) solution of agarose in 1 X TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) 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. If needed pop the air bubbles in the gel with the end of a yellow pipet tip.
B. Preparing the samples
1. While the gel is cooling, prepare the DNA samples by adding 1 uL of 6x tracking dye for each 5 uL of your sample. The tracking dyes are xylene cyanol FF (4 kB) and bromphenol blue (300 bp) or orange G (50 bp) 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 8 uL of the 1 kb ladder with 2 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 (wells will hold approimately 15 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 bromophenol blue 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.
E. Photography for room 328 imager only.
1. Place the gel on the transilluminator next to the ruler. 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 (switch on small black box and camera and switch on back of illumnator).
3. Press Quantity 1 on Computer Screen.
4. Under File choose Chemi-Doc XRS. This will cause the screen to come up showing the controls for the camera.
5. Hit the button for Step 1 and Select UV transilluminator if it is not already selected.
6. Turn on the Trans- UV Light on the gel box iteself.
7. Select Live focus and make sure the ruler is in focus by playing with iris, zoom and focus settings.
8. Once an acceptable picture is obtained press freeze.
9. To take an official picture press auto expose.
10. Save your picture onto the computer and feel free to print a copy as well if you would like.