This experiment will teach students how to prepare and load an electrophoresis gel. They will then run the gels in an electrophoresis system to separate several dyes that are of different molecular sizes and carry different charges.
This technique is fundamental to many of the procedures used in biotechnology.
Make the 0.7% agarose gel solution as follows:
To make 100 ml of gel, which is sufficient for 3 gels, weigh out 0.7 g of agarose and place into a 200- to 250-ml glass beaker or flask. Add 100 ml of 1X TBE (Tris-Borate-EDTA) buffer. Heat in the microwave for 30 seconds at a time, shaking gently each time, until the agarose is completely melted. Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. Keep warm if the class will use it within a half hour. Otherwise, allow the solution to cool and solidify. Cover and keep in the refrigerator.
Day of class:
Loosen the lid from the container of 0.7% agarose. Immerse the bottle in a distilled hot water bath to melt the liquid. Alternatively, melt the agarose in a microwave and then keep warm in a hot water bath. Do this step well ahead of time and keep the agarose warm until needed by the students. Students will need to handle the bottles so have potholders available.
After the lab, the TBE buffer solution may be poured back into the container and reused for the Restriction Digest Laboratory.
Using a Micropipet
A micropipet is a very delicate, expensive instrument that is used to dispense an extremely precise and very small volume. It is important to know the volume limits of the micropipet that you are using and to never dial either lower or higher than these limits. The volume settings on the micropipet are generally read from the top to the bottom of the number dials. Often the numbers before the decimal place will be in a different color to those that come after it. For example, on a 200-µl micropipet a volume set at 63.5 µl might have the 6 and the 3 in black lettering but the 5 might be in red lettering.
The micropipet is designed to be held in one hand. Set the micropipet to the desired volume by turning the volume adjustment knob. Using a disposable tip that is the correct size for the micropipet push the shaft of the micropipet firmly down into the sleeve of the tip until a firm seal is made.
To aspirate (pull in) the liquid, first depress the plunger to the first stop, place vertically into the liquid to a depth of about 2-3 mm and slowly release the plunger until it returns to its up position. Now you can move the micropipet out of the liquid and over to the agarose gel well or other vessel.
Place the tip against the wall of the well or other surface and begin to slowly depress the plunger, but this time you must move the plunger all the way to the second stop in order to expel all the liquid.
While still holding the plunger in the depressed position, move it out of the well or other vessel and then allow the plunger to return to its normal position. If the plunger is released while it is still in the well it will aspirate the liquid that you just deposited there!
Discard the tip into an appropriate waste container by pressing the ejector button which is found near the top of the micropipet. Always use a fresh tip for each sample to avoid cross contamination of the samples.
Tris/Borate/EDTA (TBE) buffer is commonly used in electrophoresis systems. This salt solution conducts the electric current and controls the pH of the solution during separation of DNA fragments, or in this case, dye molecules. Dilute the stock solution as necessary with distilled water to make a 1X solution.
Minerals in regular tap water will quickly stain equipment. Please rinse and air dry both the gel trays and gel boxes in distilled water. Be careful not to dislodge the wiring at the base of the gel box during this process.
When lab is complete, collect all gels in the plastic bags and dispose of in trash.
Use of Power Supplies
The power supply produces a voltage that is high enough to cause severe electrical shock if handled improperly.
Agarose is a substance derived from seaweed that forms a jelly-like matrix when dissolved in liquid. During electrophoresis an electric current is created through the agarose and molecular fragments can move through the agarose between the two electrodes.
The size of the pores in the gel and the size of the fragment trying to move will determine the rate at which each fragment progresses. The direction in which a fragment moves is determined by the charge that the fragment carries.
When using this protocol the granular dyes should be made up in the following concentrations:
For all except Orange G use 25 mg of dye, add either 10 ml of 60% glycerol solution or 4 g of sugar and make up to 10 ml with distilled water.
For Orange G use 100 mg of dye and make up as above. The Orange G is only 50 bp in size and is easily run off the end of the gel. It runs far ahead of the Bromophenol blue which is the easiest dye to see.
For the liquid dyes, if necessary, dilute with the 60% glycerol solution to the required dilution.
In this experiment you will be using electrophoresis to separate dye samples which have different sizes and charges.
STUDENTS: Check with your teacher to be sure that you understand all of the safety instructions for using this equipment.
Discard all pipette tips and gels into plastic bags that can then be placed in the regular trash. The gel box, tray and comb should be rinsed in distilled water as outlined above and then allowed to air dry. Be very careful not to damage the tiny electrode wires at each end of the gel box.
Wash your hands thoroughly and clean the area where you were working.
General information can be found at:http://ceprap.ucdavis.edu/acrobat/electrophoresis.pdf
To build your own electrophoresis chamber see:http://www.accessexcellence.org/AE/AEPC/WWC/1993/moving.html