Restriction Enzymes The ability to cut and paste

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.

Restriction Enzymes • Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites • A restriction enzyme will usually make many cuts in a DNA molecule • Yielding a set of restriction fragments • The most useful restriction enzymes cut DNA in a staggered way • Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments

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 retard the passage of molecules according to their size and shape.

The negatively charged particles move toward the positive electrode while 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. 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.