4.4 Using Gel Electrophoresis to Study Gene Molecules

Components of Gel Electrophoresis• Powdered agarose• Boiling buffer solution

Agarose Gel Concentrates

Most commonly used when separating pieces of DNA no smaller than 50 bp and no larger than 25,000 bp

Gel Stains

The gel is “run” until molecules of different sizes are thought to have completely separated.

Agarose Gel Tray. Gel trays differ depending on the manufacturer. Each has some method of sealing the ends so that liquid agarose can mold into a gel. Some gel trays, such as those made by Owl Separation Systems, make a seal with the box, so casting a gel is simple. Other trays require masking tape on the ends to make a mold. Still others, like the one shown here, have gates that screw into position: up for pouring the gel and down for running the gel.

Molecules in a Gel Box. If negatively charged molecules are loaded into the wells and run on the gel, the smaller ones run faster and farther than the larger ones toward the positive electrode. This is because smaller molecules pass more easily through the tiny spaces of the gel network.

Vocabulary

• Gel electrophoresis – a process that uses electricity to separate charged molecules, such as DNA fragments, RNA, and proteins, on a gel slab

• Agarose – a carbohydrate from seaweed that is widely used as a medium for horizontal gel electrophoresis

• Polyacrylamide – a polymer used as a gel material in vertical electrophoresis; used to separate smaller molecules, like proteins and very small pieces of DNA and RNA

• Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr

• Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color

• High through-put screening – the process of examining hundreds or thousands of samples for a particular activity

4.4 Review Questions

1. Agarose gels can be used to study what size of DNA fragments?

2. If agarose gel material is labeled 1%, what does the 1% refer to?

3. What causes molecules to be separated on an agarose gel?

4. Name two common DNA stains that are used to visualize DNA on agarose gels.

Hold micropipet

and epitubes at

eye level

Micropipet UseMicropipet Use1 Twist dial to desired volumeTwist dial to desired volume2 Add disposable pipet tipAdd disposable pipet tip3 Press plunger to first stop Press plunger to first stop 4 Insert pipet tip into solution to be Insert pipet tip into solution to be transferredtransferred5 Slowly release plunger to retrieve liquidSlowly release plunger to retrieve liquid6 Move pipet tip into desired tubeMove pipet tip into desired tube7 Press plunger past first stop to second stopPress plunger past first stop to second stop to transfer liquid, keep the plunger downto transfer liquid, keep the plunger down as you remove it from the tube.as you remove it from the tube.8. Eject tip8. Eject tip

Micropipetting

technique A

technique B

Close the tips!

Technique

C

Billiard style

Micropipet tip should

be ABOVE the well NOT IN

IT!!!!

Micropipet tip punched

right through the

gel

See dye under the

wells

NICE!