Investigative Laboratory: Generating Molecular Pedigrees Laboratory Procedure
Background
Agarose gel electrophoresis can be used to separate nucleic acids of different
lengths. In general, since nucleic acids are negatively charged, they will migrate towards
a positive electrode in a charged field. Solid agarose forms a porous matrix through
which DNA can migrate at different rates, depending on size. Large molecules will
migrate slowly through the matrix, while small molecules will migrate more quickly.
The size of a nucleic acid can be estimated by comparing its migration to that of known
size standards.
Procedure
Pouring a 1% Agarose Gel
1. In a 125 mL erlenmeyer flask add:
0.5g agarose
50 mL 1xTAE
2. Microwave for 2 min or until agarose is in solution. Swirl the flask gently,
every 15 sec., to prevent boiling over in the microwave. The gel solution may
superheat, so take care when swirling!
4. Pour molten gel into gel mold with an appropriate comb in place. Let set for at
least 30 minutes. Small air bubbles can be removed before gel starts to set using a
small pasteur pipet.
5. Fill gel buffer tank with 280 mL 1XTAE and remove gel comb
Loading and Running the Agarose Gel
1. Add 5 L of Amresco EZ Vision stain to each of your family DNA samples.
Mix briefly by pipeting up and down gently.
2. Add 1 mL of Amresco EZ Vision stain to your DNA size standard sample.
3. Load each sample in a separate well, as shown in the diagram below. Take
care not to puncture the bottom or sides of the well with the pipet tip.
Remove the tip from the well before releasing the plunger.
4. Carefully slide the top on the gel box.
5. Plug the positive lead into the red (+) terminal and the negative lead into the
black (-) terminal.
6. Turn the power supply on to 100 volts.
7. Run the gel until the blue dye has migrated down 1/2 of the length of the gel.
8. When the gel has run, turn off the power supply. NEVER
OPEN THE GEL BOX IF THE POWER IS STILL ON.
9. Remove the gel cassette (with gel) from the electrophoresis
chamber. Make sure the gel is only handled with gloved hands.
10. Place the gel on the UV Transilluminator. WARNING:
PROTECTIVE EYE WEAR MUST BE WORN AT THIS TIME!!! UV
LIGHT WILL SERIOUSLY BURN UNPROTECTED EYES!!!
11. Take a picture of the gel using the Kodak 1D Program,. Use the picture to
decide which DNA fragment is segregating with your genetic disease. This
information will be needed to solve your module mystery, and should be
presented in your posters.
12. The picture below depicts the DNA size standard that you ran on your gels.
You should use it to calculate the sizes of the two alleles on your gels.