1. Forging the Innovation Generation

2. How To Do Gel Electrophoresis
Welcome to the Project Lead the Way Virtual Academy tutorial How to Do Gel Electrophoresis.

3. Gel Electrophoresis Overview
What is gel electrophoresis?
What physics principles are used?
How is DNA electrophoresis done?
In this tutorial, you will learn * what gel electrophoresis is, * what principles of physics the process utilizes, * and how DNA electrophoresis is done.

4. What is Gel Electrophoresis?
Compares sizes:
DNA
RNA
Protein
Gel electrophoresis is a process used to separate fragments of DNA, RNA, or protein based on their size or charge.

5. What are the basic principles behind gel electrophoresis?- +
Movement in an electric field
Separation by size
Fragments of DNA or Protein in Gel Matrix
In order to tease apart the tiny particles in a sample while sorting them by size, gel electrophoresis uses two basic principles in an ingenious way. * First, an electric field is applied which causes the charged particles to move, * and second, the particles move through a gel matrix that separates them by size.

6. What are the basic principles behind gel electrophoresis?-
Movement in an electrical field
Charged particles move in an electric field
DNA fragments (-) move toward positive region -
Many molecules, including DNA, have an electrical charge in their natural state. Other molecules, for example some proteins, may be neutral, and these molecules can be made to have a charge by altering the pH or by adding ions to the buffer. *Charged molecules placed in an electric field will migrate under its influence. The charged molecules will move to the pole with the opposite charge. Remember, opposites attract. * DNA has a negative charge due to the phosphate molecules in the backbone, so DNA will migrate towards the positive region in an electrical field. +

7. What basic principles are behind gel electrophoresis?
Separation by Size
Porous gel acts like a sieve
Large particles slower than small ones
Same size = same rate = same distance
Once the particles begin moving in the electric field, they travel through a gel which helps to separate them by size. * The molecules or fragments migrate through a gel made of agarose or polyacrylamide. The gel is a porous matrix that acts like a sieve or an obstacle course for small particles. * When moving through the gel matrix, the rate of migration of each particle is related to its size. Large particles move slower than small particles. * Molecules or fragments of the same size travel at the same rate, * so particles of the same size travel the same distance. * This creates clusters, or bands, of particles that are the same length.

8. How is DNA electrophoresis done?
Power Supply
The gel electrophoresis device is a clever piece of biotechnical engineering our students will use to analyze and compare fragment sizes in DNA samples. * The device consists of a power supply and a gel box. The type of gel box will vary depending on whether DNA, RNA, or proteins are being separated.
The remainder of this lesson will focus on the electrophoresis of DNA fragments using a submarine-style gel box. It is called submarine because the gel is completely submerged under a buffer.
Submarine Gel Box

9. How is DNA electrophoresis done?
Prepare Gel
Mix proper concentration
Pour molten gel into cast
Use comb to create wells
First, a gel matrix is prepared using agarose and buffer. * The concentration of the agarose in the gel can be altered to control migration speed and improve band resolution. * The molten gel is poured into a casting tray, *and a comb is inserted to create wells, or holes, at one end for samples to be loaded. Sometimes multiple combs are added to the same gel in order to examine additional samples.
Comb
Casting tray

10. How is DNA electrophoresis done?
Remove comb
Place gel in tank
Submerge in buffer solution
Once the gel solidifies, the comb is removed * and a gel is placed into the tank, * and then the gel is submerged in a buffer solution. This buffer solution conducts the electrical current and maintains the samples at a consistent pH during electrophoresis.

11. How is DNA electrophoresis done?
Load samples
Load marker (“yardstick” for size comparisons)
* Samples are loaded into their own wells, or lanes, * and a size marker sample containing a set of known particle sizes is added to one lane. This will serve as a “yardstick” to which fragments in the other samples will be compared.

12. How is DNA electrophoresis done?
Apply electrical current to start fragment migration - +
An electrical current with a constant voltage is applied to the electrodes located at opposite ends of the gel, * so an electric field is created across the gel. * Molecules or fragments in each sample migrate through the gel, with the smallest particles traveling faster than the larger ones.

13. How is DNA electrophoresis done?
Stain the gel
Once the migration of fragments is complete, the gel is removed from its tray and stained so the fragments become visible. * After the staining process, “bands” of same-sized particles for each sample lane come into view.

14. How is DNA electrophoresis done?
marker
samples
Analyze fragment lengths
Compare bands in sample lanes to the bands in the marker lane
Same fragment lengths
The bands are analyzed to determine the lengths of DNA fragments in each sample. The relative fragment lengths in the samples can be estimated by visually comparing the relative migration distances of the unknown fragments to the migration distances of the known fragments in the marker sample. A more accurate estimate can be made by using a standard curve of the migration distances of the known fragments graphed on semi-log paper. The fragment lengths in each band in each * sample can be determined by finding an * adjacent band of known length in the marker sample. * Adjacent bands have the same fragment length.

15. References:
Bowen RA, Austgen L, and Rouge M Gel electrophoresis of DNA and RNA [Internet]. Fort Collins (CO): Colorado State University; [updated 2000 Jan 3; accessed 2008 Sep 20]. Available from:

16. Credits: Writer: Paul Legge Content Editor: Carolyn Malstrom
Production Work: CJ Amarosa