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.