Lab 5 Protocol #1:
Gel Electrophoresis of PCR Products
This protocol is similar to protocols you used for your other gels; however, there are these important differences:
1. The % agarose in the gel;
2. The volume of gel;
3. The volume of sample to load in your gel.
ALL STEPS BELOW MUST BE PERFORMED WITH LAB COATS, GOGGLES,
1. Pouring the gel: 40 ml of 0.9% agarose dissolved in 1X TAE buffer.
a. Weigh out the appropriate mass of agarose and add to 40 ml 1X TAE in a flask.
b. Loosely plug flask with paper towel and heat in microwave oven approximately 1 minute, or until agarose is fully dissolved.
c. Remove flask from microwave and allow it to cool in 55oC water bath for about 5 min (you should then be able to comfortably hold it in your gloved hands for
10 seconds); do not allow it to begin to “gel.”
d. Put comb into gel tray: the tray should be oriented in the gel box so that its ends are blocked against the sides of the box.
e. SLOWLY pour agarose/TAE mixture into gel tray to avoid creating bubbles.
f. Allow gel to solidify and cool for 20-30 min or until fully solidified.
2. Prepare samples for loading into gel
a. For each PCR sample: Remove 10 ul of your reaction and put into a new tube. Then add enough of the 6X track dye so that the track dye will be at a final concentration of 1X. Pipet up and down a couple times to mix.
b. For DNA ladder: pipette the same total volume (as samples plus dye above) of DNA ladder into a microcentrifuge tube. (The DNA ladder already has track dye in it)
c. You should have, therefore, a total of 6 samples ready to load in your gel (5 PCR samples, DNA ladder)
3. Setting up the gel
a. Reorient solidified gel in the gel box, so that comb/wells are on the appropriate end of the box (to which electrode should wells be closest?)
b. Pour 1X TAE buffer into gel box until gel it is fully covered (about 0.5 – 1 cm above gel.) c. Slowly and carefully remove comb. Remove any air bubbles with a pipette tip.
4. Loading and starting the gel
a. SLOWLY pipette each DNA + track dye sample into a separate well in the gel.
i. Pipette tip should be put down into the well, but be sure NOT to poke the pipette tip through the bottom of the well. ii. DNA ladder should be placed in a separate well, either on the left or right of samples. iii. Be sure to keep a record of which samples were loaded into which lanes.
b. Once all samples are loaded, close the gel box and attach all electrodes to the gel box and to the power pack.
i. Use color coding to be sure all electrodes are attached to the right place
(the convention is that wires to the negative electrode are black, and wires to the positive electrode are red.) ii. MAKE SURE ALL WIRES ARE FULLY CONNECTED BEFORE
TURNING ON THE POWER PACK.
c. Turn on power pack. Set on 60V or as instructed.
i. You should immediately see “bubbles” rising from the wires in the gel box ii. Within a couple minutes you should see the DNA moving into the gel from the wells.
5. Removal of gel and documentation
a. When the fastest-running dye of the gel running dye has moved about 3/4 of the way down the gel, turn off the power.
b. BE SURE POWER IS OFF AND UNPLUGED BEFORE REMOVING ANY
WIRES OR OPENING THE GEL BOX.
c. Remove wires and open gel box.
d. Carefully remove gel tray as instructed; keep gel in the tray!
e. Place gel in the staining tray with GelRed staining solution and incubate as instructed.
a. BE SURE YOU ARE WEARING LAB COAT AND GLOVES AND THAT
SHIELD OR CAMERA IS COVERING GEL BEFORE TURNING ON OR
LOOKING AT THE UV TRANSILLUMINATOR!!!
b. Place gel on the UV transilluminator
c. Turn on UV transilluminator. GelRed bound to DNA will fluoresce indicating bands of DNA.
d. Photograph gel with the camera as instructed.
e. Dispose of gel in the appropriate waste receptacle as instructed.
f. Discard all solid waste as