Preparing an Agarose Gel

DNA gels are made of agarose, a highly purified agar, which is heated and dissolved in a buffer solution. The agarose molecules form a matrix with pores between them. The more concentrated the agarose, the smaller the pores. We will be using a 0.8% agarose gel (there are 0.8 grams of agarose per 100 mL of buffer) because we are looking at large DNA fragments thousands of base pairs in length. A 2% agarose gel separates DNA fragments which differ in length by as few as 50-100 base pairs.

Before beginning, double check the size of the gel you are preparing. All volumes and weights are given for a 50 mL gel.

Weigh 0.4 grams of agarose powder and place it in a 125 or 250 mL flask.
Add 50 mL of 1X TBE buffer to the flask. Swirl to mix the solution.
Place the flask in the microwave. Heat on high until the solution is completely clear and no small floating particles are visible (about 2 minutes). Swirl the flask frequently to mix the solution and prevent the agarose from burning.
* Do not allow the agarose to boil over.
* Use hot mitts when handling the flask because it will be very hot.
Cool the solution to 55 C before pouring the gel into the plastic casting tray. Higher temperatures will melt the plastic tray.
While the mixture cools, tape the ends of the gel tray with masking tape. Place the plastic comb in the slots on the side of the gel tray. The comb teeth should not touch the bottom of the tray.
Pour the agarose mixture into the gel tray until the comb teeth are immersed about 6 mm or 1/4" into the agarose. Push any bubbles to the side farthest from the wells.
Allow the agarose gel to cool until solidified. The gel will appear a cloudy white color and will feel cool to the touch (about 20 minutes). Gels can be stored, wrapped in plastic wrap, in the refrigerator for a few days.

Loading and Running an Agarose Gel

Gel electrophoresis is an important molecular biology tool. DNA sequencing, fingerprinting (or "profiling"), and genetic engineering are based upon it. Gel electrophoresis separates DNA fragments by their size or molecular weight. The agarose gel acts like a sieve, separating different sized fragments while the electric current provides the driving force. DNA, a negatively charge molecule, is attracted to the positive electrode; the voltage determines how fast the DNA will travel through the gel. Larger molecules or DNA fragments become entangled in the gel and travel more slowly, while smaller ones pass through more easily and travel farther down the gel. Similar-sized DNA fragments travel at the same rate and form a tight bunch called a "band". The DNA in the gel must be stained in order to see the bands.

Remove the comb from the wells by pulling straight up on the comb. Be careful not to tear the wells as you remove the comb. Remove the tape from both ends of the gel tray.
Place the gel tray in the gel box with the wells closest to the negative (black) electrode.
Add enough 1X TBE buffer to fill the buffer tank and submerge the gel about 1/4 inch.
On a piece of wax paper, mix 5 uL of gel loading dye with 10 uL of your DNA sample. Mix the solution by pipetting the dye up and down into the 10 uL DNA sample. The gel loading dye contains glycerol which will make your DNA more dense so that it will sink into the wells. It also contains dye molecules which are smaller and travel faster through the gel than the DNA molecules. The dye molecules provide a visual tracking method so you know how far the DNA has traveled through the gel.
Rinse the pipette or capillary tube with clean water by gently pipetting up and down two or three times.
Make a sketch of the gel; include all of the wells and the positive (red) and negative (black) electrodes. Indicate under each well the name of the sample to be loaded.
Pipette 10 uL of the first sample, usually a reference sample such as a 1 kb ladder, into the first well. Keep the tip of the pipettor ABOVE the well. The DNA will sink into the well because it has been mixed with loading dye. If you puncture the bottom of the well your DNA run out the bottom of the gel into the buffer tank.
* Molecular biologists often use a size standard called a 1 kb DNA ladder. The DNA ladder produces several different sized fragments or bands and can be used to estimate the size of an unknown DNA fragment.
Rinse the pipette or capillary tube in the buffer tank by gently pipetting up and down two or three times.
You are now ready to load the next sample into the next well. Repeat steps #7-8 until all of the samples and controls have been loaded into the gel. Your teacher will tell you how many lanes to save for concentration standards and which ones to load. Remember to record on your sketch the order the samples were loaded (including standards!)
* A concentration standard is used to estimate how much DNA is present in a sample.
Close the top of the box. Plug the leads into the gel box. The black lead is the negative lead and should be plugged in closest to the wells. The red lead is the positive lead and should be plugged in furthest from the wells.
* Remember: Red ahead.
Plug the other end of the leads into the power source and turn it on. Run the gel at between 80-120 volts until the loading dye has traveled 1/2 - 3/4 of the way down the gel (about 30-45 minutes).
Turn off the power supply. Unplug the leads and the power supply before opening the gel box.
The gels may be wrapped in Syran Wrap and stored overnight until they can be stained.

Staining the DNA

The DNA must be stained in order to be seen. DNA can be stained with fluorescent or chemical dyes. Research laboratories use ethidium bromide, an ultraviolet (UV) fluorescent stain, because it shows very small amounts of DNA and is faster to use. Ethidium bromide, however, can cause cancer and mutate DNA. We will use methylene blue, a chemical dye, which binds to DNA. Methylene blue may stain your hands and clothes if you spill it, but it is not toxic.
Hint: Wear gloves when working with methylene blue.

Place the gel in a petri-dish or plastic container which is a little larger than the gel. Add enough 0.025% methylene blue solution to cover the gel about 1/4". Stain the gel for 20-30 minutes.
Carefully pour off as much of the methylene blue solution as possible into a small beaker. Your entire gel will appear deep blue.
Hint: Save the methlyene blue solution; it may be reused many times.
Rinse the gel in running tap water. Let gel soak covered with water for 10 minutes. Rock the tray occasionally to help destain the gel. Repeat 3-4 times. The DNA bands will become more distinct as the gel destains.
Hint: If necessary, you may continue to destain the gel overnight in a small amount of water; the gel will destain too much if left in a large amount of water. Cover the staining tray to prevent evaporation.
Draw the bands on your gel sketch; use rulers to ensure the proper location of the bands.
You may choose to photograph/photocopy your gel or view it on the overhead projector. Gels can be stored in plastic zip lock bags in the refrigerator.

on Plans

Student Activity:

DNA Analysis from Cheek Cells

Agarose Gel Preparation

Harri's Hawks: Case Study

Materials List

Resource List

The University of Arizona
Department of Biochemistry and Molecular Biophysics
General Biology Program for Secondary Teachers
warder@email.arizona.edu