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
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
- 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
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
DNA Analysis from Cheek Cells
Agarose Gel Preparation
Harri's Hawks: Case Study
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