Click here to know where to purchase electrophoresis units, reagents, standards, dyes, precasted gels and all you may need to perform an electrophoresis. In your first run you may run only your standards and once you confirmed that your system is operating correctly you may run your samples together with the standards.
First, read about electrophoresis units components.
Now you are ready to start preparing the gel.
1) Seal the open borders of a clean and dry glass plate with tape
for forming a mold.
2) Prepare an adequate volume of electrophoresis buffer (TBE:
Tris/borate/EDTA or TAE: Tris/acetate/EDTA) to fill the electrophoresis
tank and to prepare the gel.
3) Add the needed amount of powdered agarose (typically 8-15%) to the electrophoresis buffer (TBE or TAE) in an Erlenmeyer flask. The buffer should not occupy more than 50% of the volume of the flask.
4) Heat the agarose solution in a microwave oven or in water bath to allow all of the grains of agarose to dissolve. If part of the buffer evaporated during the heating, bring the solution back to the original volume through the addition of buffer.
5) Cool down the solution to 60 C. You may add ethidium bromide (Et Br) (10 mg/l in water to a final concentration of 0.5 um/ml) in this step or you can submerge the gel in an Et Br solution once it solidifies.
6) Position the comb 0.5-1.0 mm above the plate, thus permitting that a complete well is formed when the agarose solidifies. Air bubbles under or between the teeth of the comb should be avoided. Using a Pasteur pipette seal the glass plate with small amounts of agarose solution. Once the seals are set, pour the gel in the glass plate.
7) Remove the comb and the tape when the gel has completely hardened (30 to 40 minutes at room temperature) and place the gel in the electrophoresis tank. Add enough electrophoresis buffer to the tank to cover the gel (about 1 mm of depth). The top of the wells should be submerged.
8) Mix the samples and standards with appropriate amounts of loading buffer (10x). Slowly load the mixture into the wells with a micropipette. Avoid any mix of the samples between wells.
10) If the unit is working you should see bubbles that are formed in the buffer due to the electrical field that has been creates; and later you should see the dyes running in the gel.
11)If ethidium bromide was present in the agarose gel,
DNA may be photographed by with UV
light (>500uW/cm2). The location of DNA within
the gel and may be determined by a film image of light transmitted by a
fluorescing DNA. Thus, a wide range of bands or fragments of DNA
can be detected on the film (from 200 bp to ~ 50 kb in length). Size and
amount of DNA can be calculated running standards of known size and concentration.
DNA bands may be recovered from the gel and utilized for a diversity
of cloning designs. See Current Protocols on Molecular Biology (1995, John
Wiley & Sons Inc., Virginia Benson, Canada) for more information on
cloning and DNA analysis protocols.
