Gel Electrophoresis What is Gel Electrophoresis? Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to to retard the passage of molecules according to their size and shape. BIOTECHNOLOGY One of the basic tools of modern biotechnology is gene splicing. This is the process of removing a functional DNA fragment ( a gene) from one organism and combining it with the DNA of another organism to study how the gene works. The desired result is to have the new organisms carry out the expression of the gene that has been inserted. Restriction Enzymes The ability to cut and paste DNA predictably is due to the use of restriction enzymes. They were first identified in and isolated from the bacteria that use them as a natural defense mechanism to cut up the invading DNA of bacteriophages – viruses that infect bacteria. They are named for the The negatively charged particles move toward the positive electrode while the the positive charge particles move toward the negative electrode. How does electrophoresis work? • The gel is made from agar • DNA is a negative molecules • Molecules sort based on •Charge •Size •shape What is agar? Agar comes from sea weed. What is it used for? The gel is 1% agarous and has no electrical charge. How does it work? • DNA is cut into smaller fragments. • Loading dye is used to indicate the fragments of DNA are behind the dye • The negative DNA molecule is attracted to the positive electrode. •The smallest fragments move the greatest distance. Procedure Remove comb and observe wells. Place carbon paper in each end of the tray. Cover with buffer, making sure the allow buffer to overflow into each end of the tray. Load gels. Connect the electrodes. Turn on power supply. Allow gels to run – make sure you see bubbles coming from the electrodes. PROCEDURE (CONTINUED) It will take about 30 minutes for the gel to run. Turn off power supply and remove electrodes. Pour off buffer into the designated container. Carefully remove gel from gel box and place in glad container and cover with stain. Store in appropriate location. What is significant about the bubbles? They indicate that electrolysis of water is taking place. One electrode will have a lot of bubbles and the other will have a lesser amount. Why the difference? The formula for water is H2O and the splitting of the molecule will produce twice as many atoms of hydrogen.